Bioactive compositions from Theacea plants and processes for their production and use

ABSTRACT

The present invention relates to isolated bioactive compositions containing bioactive fractions derived from Theacea plants. The present invention also relates to bioactive topical formulations containing the bioactive compositions. The present invention further relates to methods of using the bioactive compositions of the present invention, including, for example, methods for inhibiting inflammatory activity in skin tissue of a mammal, for protecting skin tissue of a mammal from ultraviolet light-induced damage, and for normalizing skin disorders in skin tissue of a mammal. The present invention also relates to methods for isolating bioactive fractions derived from cell juice or a cell walls component a Theacea plant.

This application is a divisional of U.S. patent application Ser. No.12/335,534, filed Dec. 15, 2008, issued as U.S. Pat. No. 8,043,635 onOct. 25, 2011, which is a divisional of U.S. patent application Ser. No.11/033,925, filed Jan. 12, 2005, issued as U.S. Pat. No. 7,473,435 onJan. 6, 2009, which claims the priority benefit of U.S. ProvisionalPatent Application Ser. No. 60/535,861, filed Jan. 12, 2004, thedisclosures of which are hereby incorporated herein by reference intheir entirety.

FIELD OF THE INVENTION

The present invention relates to bioactive compositions, processes fortheir production from Theacea plants, and uses of these compositions.

BACKGROUND OF THE INVENTION

The Theacea (tea plants) family includes trees or shrubs comprisingabout 40 genera and 600 species. Camellia sinensis occupies a uniqueposition in the Theacea family, because this particular species of plantis predominantly used as a single raw material source to produce allthree basic kinds of tea: green tea, oolong tea, and black tea(collectively referred to herein as the “tea plant”). According to somesources, there is a fourth type of tea, i.e., the so-called “white tea,”which is produced exclusively from the buds or tips of the tea plant.

The three basic forms of tea are determined by the degree of processing,which involves the identical tender young tea leaves. The leaves areplucked, sorted, cleaned, and variously oxidized before steaming ordrying. The term “fermentation” is frequently used to describe theprocessing of tea, but the term “oxidation” is a much more accuratedescription of the chemical transformations which take place.

Although there are some variations in the processing, it is generallyagreed that green tea has the lowest degree of oxidation and that blacktea has the highest. Oolong tea is considered to be partially oxidized,and thus occupies the place between green and black tea. With respect toprocessing, there is very little difference (or no difference at all)between green and white tea.

Green tea is made from fresh leaves that are steamed and wilted, andthen immediately dried. Black tea is made from leaves that are wiltedand crushed in rollers, then allowed to oxidize for several hours beforethey are dried. Oolong tea comes from leaves that are only partiallyoxidized before drying.

Worldwide, tea is the second (after water) most commonly consumedliquid, and is the sixth (after water, soft drinks, coffee, beer, andmilk) most commonly consumed liquid in the United States. Teaconsumption continues to increase worldwide, especially due to thegrowing public awareness concerning health benefits of this liquid.There is a growing number of publications suggesting anti-angiogenic,anti-bacterial, anti-cancerogenic, anti-inflammatory, anti-mutagenic,anti-oxidant, anti-septic, and detoxifying properties of teas and theiringredients. The list of tea benefits also includes reduction of therisk of rheumatoid arthritis, lowering cholesterol levels, andanti-diabetic properties. Not all of these benefits have been proven tobe statistically significant. Nevertheless, the very broad spectrum oftea benefits reflects the unique composition of the very powerfulbiologically active substances, which exist in fresh plant leaves andsurvive conventional tea processing.

In particular, fresh leaves of Camellia sinensis have been reported tocontain 22.2% polyphenols, 17.2% protein, 4.3% caffeine, 27.0% crudefiber, 0.5% starch, 3.5% reducing sugars, 6.5% pectins, 2.0% etherextract, and 5.6% ash (Duke, J. A., Handbook of Energy Crops (1983), seewww.hort.purdue.edu/newcrop/duke_energy/Camellia _(—) sinensis.html).Per 100 g, the leaf is reported to contain 8.0 g H₂O, 24.5 g protein,2.8 g fat, 58.8 g total carbohydrate, 8.7 g fiber, 5.9 g ash, 327 mg Ca,313 mg P, 24.3 mg Fe, 50 mg Na, 2700 μg β-carotene equivalent, 0.07 mgthiamine, 0.8 mg riboflavin, 7.6 mg niacin, and 9 mg ascorbic acid.Another report tallies 8.0 g H₂O, 28.3 g protein, 4.8 g fat, 53.6 gtotal carbohydrate, 9.6 g fiber, 5.6 g ash, 245 mg Ca, 415 mg P, 18.9 mgFe, 60 mg Na, 8400 μg β-carotene equivalent, 0.38 mg thiamine, 1.24 mgriboflavin, 4.6 mg niacin, and 230 mg ascorbic acid. Yet another gives8.1 g H₂O, 24.1 g protein, 3.5 g fat, 59.0 g total carbohydrate, 9.7 gfiber, 5.3 g ash, 320 mg Ca, 185 mg P, 31.6 mg Fe, 8400 μg β-caroteneequivalent, 0.07 mg thiamine, 0.79 mg riboflavin, 7.3 mg niacin, and 85mg ascorbic acid (J. A. Duke and A. A. Atchley, “Proximate Analysis,”In: Christie, B. R. (ed.), The Handbook of Plant Science in Agriculture,CRC Press, Inc., Boca Raton, Fla. (1984)).

Leaves also contain carotene, riboflavin, nicotinic acid, pantothenicacid, and ascorbic acid. Caffeine and tannin are among the more activeconstituents (Council for Scientific and Industrial Research,1948-1976). Ascorbic acid, present in the fresh leaf, is destroyed inmaking black tea. Malic and oxatic acids occur, along with kaempferol,quercitrin, theophylline, theobromine, xanthine, hypoxanthine, adenine,gums, dextrins, and inositol. Chief components of the volatile oil(0.007-0.014% fresh weight of leaves) are hexenal, hexenol, and loweraldehydes, butyraldehyde, isobuteraldehyde, isovaleraldehyde, as well asn-hexyl, benzyl and phenylethyl alcohols, phenols, cresol, hexoic acid,n-octyl alcohol, geraniol, linalool, acetophenone, benzyl alcohol, andcitral.

It was found that the fresh tea leaf has an unusually high level offlavanol group of polyphenols (catechins), which may reach up to 30% ofleaf dry matter. Catechins include predominantly (−)-epicatechin,(−)-epicatechin gallate, (−)-epigallocatechin and (−)-epigallocatechingallate. Additionally there are unique to tea 3-galloylquinic acid(theogallin) and unique amino acid theanine (5-N-ethylglutamine) (Duke,J. A., Handbook of Energy Crops (1983), seewww.hort.purdue.edu/newcrop/duke_energy/Camellia _(—) sinensis.html).

Tea leaves contain high levels of polyphenol-oxidase and peroxidase. Thefirst enzyme catalyzes the aerobic oxidation of the catechins and thisprocess is initiated when the integrity of the leaf cell structure isdisrupted. Phenol-oxidase is responsible for generation of bisflavanols,theaflavins, epitheaflavic acids, and thearubigens, which constitute thelargest mass of the extractable matter in black tea. Most of thesecompounds readily form complexes with caffeine, which has significantlevel (2-4% of dry matter) in fresh leaves. Peroxidase plays importantrole in generation of the above complexes with proanthocyanidins. Thecatechin quinones also initiate the formation of many of the hundreds ofvolatile compounds found in the black tea aroma fraction. Additionally,the transformation of relatively soluble glycosides to lower solubilityaglycones takes place.

All complex cascades of the above processes are initiated by disruptionof the leaf cell structure and are intensified with the time ofoxidation. As result, the composition of black tea, which is usuallyprocessed with intensive rolling or cutting and relatively long timeoxidation, is much more different than that of the fresh leaf. Althoughgreen tea (and white tea) is processed with minimum oxidation, and itscomposition more similar to that of fresh leaves, there arenon-enzymatic and enzymatically catalyzed changes, which occur extremelyrapidly following plucking, and new volatile substances that areproduced during the drying stage. Thus, even relatively gentle green teaprocessing initiates certain departure from original fresh plantcomposition and can diminish the therapeutic value and other potentialbenefits of fresh tea plant leaves.

Numerous recent studies clearly demonstrate that therapeutic benefits oftea are decreased in the following sequence: white tea>green tea>oolongtea>black tea. Thus, exploration of fresh tea plants may prevent thedegradation of specific activities, which are observed as a result ofconventional tea processing. Fresh, tender Camellia leaves containapproximately 80% water. Swelling and dehydration of the cells isprevented by the cells' rigid cell walls. The disruption of the cellwall structure triggers the dehydration of fresh plant tissue followedby the sequence of unwanted physico-chemical and biochemical processes:osmotic shock, decompartmentalization and disruption of enzymes,hydrolysis and oxidation, polymerization of phenols, transformation ofglycosides to aglycones, generation of products of Maillard reaction,isomerization, and microbial contamination. Therefore, fresh Camelliacontains very broad spectrum of biologically active substances and onlypart of them became available during conventional extraction processes.Thus, only cell walls, catabolites, and stable metabolites can beextracted with boiled water to obtain tea drink or for extraction withdifferent solvents to obtain limited parts of biologically activecomponents (predominantly polyphenols and flavonoids).

In light of the potential of fresh tea leaves as sources of valuabletherapeutic and other potentially beneficial bioactive compositions,exploration of fresh tea plants is needed to determine how to maximizetheir therapeutic and other potentially beneficial bioactive properties.

SUMMARY OF THE INVENTION

The present invention relates to a bioactive composition. In oneembodiment, the bioactive composition includes an isolated bioactivefraction derived from a Theacea plant. Suitable bioactive fractions caninclude, without limitation, a cell walls fraction, a cell wallsfraction extract, a membrane fraction, a membrane fraction extract, acytoplasm fraction, a cytoplasm fraction extract, a cell juice serum,and/or combinations thereof.

The present invention also relates to a bioactive topical formulationsuitable for topical application to a mammal. In one embodiment, thebioactive topical formulation includes a topically effective amount ofthe bioactive composition of the present invention. The bioactivetopical formulation can further include a topically acceptable carrier.

The present invention also relates to a method for inhibitinginflammatory activity in skin tissue of a mammal. This method involvesproviding the bioactive composition according to the present invention.The method further involves applying the bioactive composition to theskin tissue in an amount effective to inhibit inflammatory activity inthe skin tissue.

The present invention also relates to a method of protecting skin tissueof a mammal from ultraviolet light-induced damage. This method involvesproviding the bioactive composition of the present invention. The methodfurther involves applying the bioactive composition to the skin tissuein an amount effective to reduce ultraviolet light-induced damage of theskin tissue and to prevent oxidative damage of the skin tissue.

The present invention also relates to a method for normalizing skindisorders in skin tissue of a mammal. This method involves providing thebioactive composition of the present invention. The method furtherinvolves applying the bioactive composition to the skin tissue in anamount effective to normalize a cell disorder in the skin tissue.

The present invention also relates to a method for isolating a bioactivefraction derived from cell juice of a Theacea plant. This methodinvolves providing a Theacea plant. The Theacea plant is then separatedinto cell juice and a cell walls component. The cell juice is thentreated under conditions effective to yield a bioactive fraction.Suitable bioactive fractions include, without limitation, a membranefraction, a membrane fraction extract, a cytoplasm fraction, a cytoplasmfraction extract, and/or a cell juice serum. The bioactive fraction isthen isolated from the treated cell juice. The present invention furtherrelates to an isolated bioactive composition produced by this method.

The present invention also relates to a method for isolating a bioactivefraction derived from a cell walls component of a Theacea plant. Thismethod involves providing a Theacea plant. The Theacea plant is thenseparated into cell juice and a cell walls component. The cell wallscomponent is treated under conditions effective to yield a bioactivefraction. The bioactive fraction is then isolated from the treated cellwalls component. The present invention further relates to an isolatedbioactive fraction produced by this method.

The present invention is useful in addressing the deficiencies ofconventional tea processing methods, particularly the inability ofconventional tea processing to preserve a broad spectrum of potentbioactive compositions. As provided by the present invention, processingof fresh Camellia biomass without fermentation and excessive heattreatment can yield more powerful and diversified bioactive compositionsthan products of conventional tea processing.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic drawing demonstrating one embodiment of theprocess for preparing the bioactive compositions of the presentinvention.

FIG. 2 is a graph showing the UV/VIS spectra of extracts of cell wallsfraction and conventional teas (Dilution 1:1000).

FIG. 3 is a graph showing the UV/VIS spectra of Camellia bioactivecompositions (Dilution 1:4000).

FIG. 4 is a graph showing the absorbance spectra of extracts of cellwalls fraction and conventional teas applied on Vitro-Skin® testingsubstrate (IMS Testing Group, Milford, Conn.). Dry matter levels areequalized.

FIG. 5 is a graph showing the absorbance spectra of Camellia bioactivecompositions applied on Vitro-Skin® testing substrate (IMS TestingGroup, Milford, Conn.). Dry matter levels are equalized.

FIG. 6 is a graph showing the absorbance spectra of Camellia bioactivecompositions and white tea extract applied on Vitro-Skin® testingsubstrate (IMS Testing Group, Milford, Conn.).

FIG. 7A is a graph showing the absorbance spectra of Camellia membranefraction extract in diluted solution (1:200) and applied on Vitro-Skin®testing substrate (IMS Testing Group, Milford, Conn.). FIG. 7B is agraph showing the absorbance spectra of Camellia cell juice serum indiluted solution (1:200) and applied on Vitro-Skin® testing substrate(IMS Testing Group, Milford, Conn.).

FIG. 8A is a graph showing the absorbance spectra of Barley (Hordeumvulgare) cell juice serum in diluted solution (1:200) and applied onVitro-Skin® testing substrate (IMS Testing Group, Milford, Conn.). FIG.8B is a graph showing the absorbance spectra of Sage (Salviaofficinalis) cell juice serum in diluted solution (1:200) and applied onVitro-Skin® testing substrate (IMS Testing Group, Milford, Conn.).

FIG. 9 is a graph showing the effect of broad spectrum UV irradiation onVitro-Skin® testing substrate (IMS Testing Group, Milford, Conn.).

FIG. 10 is a graph showing the effect of broad spectrum UV irradiationon white tea extract applied on Vitro-Skin® testing substrate (IMSTesting Group, Milford, Conn.).

FIG. 11 is a graph showing the effect of broad spectrum UV irradiationon cell walls fraction extract applied on Vitro-Skin® testing substrate(IMS Testing Group, Milford, Conn.).

FIG. 12 is a graph showing the effect of broad spectrum UV radiation onCamellia membrane fraction extract applied on Vitro-Skin® testingsubstrate (IMS Testing Group, Milford, Conn.).

FIG. 13 is a graph showing the effect of broad spectrum UV radiation onCamellia cell juice serum applied on Vitro-Skin® testing substrate (IMSTesting Group, Milford, Conn.).

FIG. 14 is a graph showing the effect of white tea extract onMDA-MB-435S cells cultivated for 24 hours and 48 hours.

FIG. 15 is a graph showing the effect of white tea extract on MCF-7cells cultivated for 24 hours (control) and for 24 hours and 48 hours inthe presence of 5 ng/ml TGF-β.

FIG. 16 is a graph showing the effect of cell walls fraction extract onMDA-MB-435S cells cultivated for 24 hours and 48 hours.

FIG. 17 is a graph showing the effect of cell walls fraction extract onMCF-7 cells cultivated for 24 hours (control) and for 24 hours and 48hours in the presence of 5 ng/ml TGF-β.

FIG. 18 is a graph showing the effect of membrane fraction extract onMDA-MB-435S cells cultivated for 24 hours and 48 hours.

FIG. 19 is a graph showing the effect of membrane fraction extract onMCF-7 cells cultivated for 24 hours (control) and for 24 hours and 48hours in the presence of 5 ng/ml TGF-β.

FIG. 20 is a graph showing the effect of cell juice serum on MDA-MB-435Scells cultivated for 24 hours and 48 hours.

FIG. 21 is a graph showing the effect of cell juice serum on MCF-7 cellscultivated for 24 hours (control) and for 24 hours and 48 hours in thepresence of 5 ng/ml TGF-β.

FIG. 22 is a graph showing the effect of white tea extract on Mono Mac 6cells cultivated for 24 and 48 hours.

FIG. 23 is a graph showing the effect of white tea extract on Mono Mac 6cells cultivated for 24 hours and 48 hours in the presence of 10 nM PMA.

FIG. 24 is a graph showing the effect of cell walls fraction extract onMono Mac 6 cells cultivated for 24 hours and 48 hours.

FIG. 25 is a graph showing the effect of cell walls fraction extract onMono Mac 6 cells cultivated for 24 hours and 48 hours in the presence of10 nM PMA.

FIG. 26 is a graph showing the effect of membrane fraction extract onMono Mac 6 cells cultivated for 24 hours and 48 hours.

FIG. 27 is a graph showing the effect of membrane fraction extract onMono Mac 6 cells cultivated for 24 hours and 48 hours in the presence of10 nM PMA.

FIG. 28 is a graph showing the effect of cell juice serum on Mono Mac 6cells cultivated for 24 hours and 48 hours.

FIG. 29 is a graph showing the effect of cell juice serum on Mono Mac 6cells cultivated for 24 hours and 48 hours in the presence of 10 nM PMA.

FIG. 30 is a graph showing the effect of white tea extract on level ofMMPs secreted by PMA stimulated Mono Mac 6 cells.

FIG. 31 is a graph showing the effect of cell walls fraction extract onlevel of MMPs secreted by PMA stimulated Mono Mac 6 cells.

FIG. 32 is a graph showing the effect of membrane fraction extract onlevel of MMPs secreted by PMA stimulated Mono Mac 6 cells.

FIG. 33 is a graph showing the effect of cell juice serum on level ofMMPs secreted by PMA stimulated Mono Mac 6 cells.

FIG. 34 is a gelatin zymogram of culture media collected after 48 hoursexposure of Mono Mac 6 cells to white tea extract, along with culturemedia collected from cells cultured in the absence (U) or presence (S)of 10 nM PMA, but in the absence of the Camellia compositions.

FIG. 35 is a gelatin zymogram of culture media collected after 48 hoursexposure of Mono Mac 6 cells to cell walls fraction extract, along withculture media collected from cells cultured in the absence (U) orpresence (S) of 10 nM PMA, but in the absence of the Camelliacompositions.

FIG. 36 is a gelatin zymogram of culture media collected after 48 hoursexposure of Mono Mac 6 cells to membrane fraction extract, along withculture media collected from cells cultured in the absence (U) orpresence (S) of 10 nM PMA, but in the absence of the Camelliacompositions.

FIG. 37 is a gelatin zymogram of culture media collected after 48 hoursexposure of Mono Mac 6 cells to cell juice serum, along with culturemedia collected from cells cultured in the absence (U) or presence (S)of 10 nM PMA, but in the absence of the Camellia compositions.

FIG. 38 is a bar graph comparing the content of various catechins in thewhite tea extract (“WTE”) and in the cell walls fraction extract(“CWFE”), the membrane fraction extract (“MFE”), and the cell juiceserum (“CJS”) of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a bioactive composition. In oneembodiment, the bioactive composition includes an isolated bioactivefraction derived from a Theacea plant. As used herein, the term“isolated bioactive fraction” is meant to include fractions that areisolated from a Theacea plant (e.g., fresh biomass of a Theacea plant)that has not undergone any conventional tea processing (e.g., heattreatment, oxidation, fermentation, drying). Suitable isolated bioactivefractions can include, without limitation, a cell walls fraction, a cellwalls fraction extract, a membrane fraction, a membrane fractionextract, a cytoplasm fraction, a cytoplasm fraction extract, a celljuice serum, and/or combinations thereof.

The bioactive compositions and bioactive fractions of the presentinvention can have various catechin profiles and total catechin contentamounts, as defined below, and as determined using conventional catechindiagnostic methods well known in the art. As used herein, the term“catechin” generally refers to all catechins, including, but not limitedto, the following specific types of catechins: (i) (−)-epigallocatechin(see CAS No. 970-74-1, which is hereby incorporated by reference in itsentirety); (ii) (+)-catechin (see CAS No. 7295-85-4, which is herebyincorporated by reference in its entirety); (iii) (−)-epicatechin (seeCAS No. 490-46-0, which is hereby incorporated by reference in itsentirety); (iv) (−)-epigallocatechin gallate (see CAS No. 989-51-5,which is hereby incorporated by reference in its entirety); (v)(−)-gallocatechin gallate (see CAS No. 4233-96-9, which is herebyincorporated by reference in its entirety); and (vi) (−)-epicatechingallate (see CAS No. 1257-08-5, which is hereby incorporated byreference in its entirety). “Total catechin content” (as used herein)refers to the combined content level of all catechins contained in aparticular bioactive composition or bioactive fraction of the presentinvention, and is not meant to be limited to the content levels of justthe specific types of catechins listed herein above. As used herein, theterm “catechin content profile” is used to describe the amounts ofselected catechins contained in a particular bioactive composition orbioactive fraction of the present invention.

In one embodiment of the bioactive composition of the present invention,the bioactive fraction can be a cell walls fraction.

In one embodiment of the bioactive composition of the present invention,the bioactive fraction can be a cell walls fraction extract. In aspecific embodiment of the present invention, the cell walls fractionextract can have a total catechin content of between about 2.1 and about4.5 milligrams per gram of dry matter, particularly between about 2.6and about 4.0 milligrams per gram of dry matter, and more particularlybetween about 3.0 and about 3.6 milligrams per gram of dry matter. Inanother specific embodiment, the cell walls fraction extract can have acatechin content profile as follows: (i) between about 2.0 and about 3.0milligrams of (+)-catechin per gram of dry matter of the cell wallsfraction extract; (ii) between about 0.005 and about 0.02 milligrams of(−)-epicatechin per gram of dry matter of the cell walls fractionextract; (iii) between about 0.005 and about 0.02 milligrams of(−)-epigallocatechin gallate per gram of dry matter of the cell wallsfraction extract; and (iv) between about 0.003 and about 0.01 milligramsof (−)-epicatechin gallate per gram of dry matter of the cell wallsfraction extract. More particularly, the cell walls fraction extract canhave a catechin content profile as follows: (i) between about 2.2 andabout 2.7 milligrams of (+)-catechin per gram of dry matter of the cellwalls fraction extract; (ii) between about 0.01 and about 0.015milligrams of (−)-epicatechin per gram of dry matter of the cell wallsfraction extract; (iii) between about 0.01 and about 0.015 milligrams of(−)-epigallocatechin gallate per gram of dry matter of the cell wallsfraction extract; and (iv) between about 0.005 and about 0.007milligrams of (−)-epicatechin gallate per gram of dry matter of the cellwalls fraction extract.

In one embodiment of the bioactive composition of the present invention,the bioactive fraction can be a membrane fraction.

In one embodiment of the bioactive composition of the present invention,the bioactive fraction can be a membrane fraction extract. In a specificembodiment of the present invention, the membrane fraction extract canhave a total catechin content of between about 15.0 and about 30.5milligrams per gram of dry matter, particularly between about 18.0 andabout 27.5 milligrams per gram of dry matter, and more particularlybetween about 21.0 and about 24.5 milligrams per gram of dry matter. Inanother specific embodiment, the membrane fraction extract can have acatechin content profile as follows: (i) between about 1.7 and about 3.3milligrams of (−)-epigallocatechin per gram of dry matter of themembrane fraction extract; (ii) between about 6.1 and about 10.2milligrams of (+)-catechin per gram of dry matter of the membranefraction extract; (iii) between about 0.3 and about 1.1 milligrams of(−)-epicatechin per gram of dry matter of the membrane fraction extract;(iv) between about 6.2 and about 12.5 milligrams of (−)-epigallocatechingallate per gram of dry matter of the membrane fraction extract; (v)between about 0.007 and about 0.03 milligrams of (−)-gallocatechingallate per gram of dry matter of the membrane fraction extract; and(vi) between about 1.3 and about 3.3 milligrams of (−)-epicatechingallate per gram of dry matter of the membrane fraction extract. Moreparticularly, the membrane fraction extract can have a catechin contentprofile as follows: (i) between about 2.0 and about 3.0 milligrams of(−)-epigallocatechin per gram of dry matter of the membrane fractionextract; (ii) between about 7.0 and about 9.0 milligrams of (+)-catechinper gram of dry matter of the membrane fraction extract; (iii) betweenabout 0.5 and about 0.9 milligrams of (−)-epicatechin per gram of drymatter of the membrane fraction extract; (iv) between about 8.0 andabout 10.0 milligrams of (−)-epigallocatechin gallate per gram of drymatter of the membrane fraction extract; (v) between about 0.01 andabout 0.02 milligrams of (−)-gallocatechin gallate per gram of drymatter of the membrane fraction extract; and (vi) between about 1.8 andabout 2.8 milligrams of (−)-epicatechin gallate per gram of dry matterof the membrane fraction extract.

In one embodiment of the bioactive composition of the present invention,the bioactive fraction can be a cytoplasm fraction.

In one embodiment of the bioactive composition of the present invention,the bioactive fraction can be a cytoplasm fraction extract.

In one embodiment of the bioactive composition of the present invention,the bioactive fraction can be a cell juice serum. In a specificembodiment, the cell juice serum can have a total catechin content ofbetween about 8.0 and about 20.0 milligrams per gram of dry matter,particularly between about 10.0 and about 18.0 milligrams per gram ofdry matter, and more particularly between about 12.0 and about 16.0milligrams per gram of dry matter. In another specific embodiment, thecell juice serum can have a catechin content profile as follows: (i)between about 2.1 and about 4.4 milligrams of (−)-epigallocatechin pergram of dry matter of the cell juice serum; (ii) between about 4.2 andabout 8.6 milligrams of (+)-catechin per gram of dry matter of the celljuice serum; (iii) between about 0.2 and about 2.0 milligrams of(−)-epicatechin per gram of dry matter of the cell juice serum; (iv)between about 1.2 and about 3.2 milligrams of (−)-epigallocatechingallate per gram of dry matter of the cell juice serum; (v) betweenabout 0.01 and about 0.1 milligrams of (−)-gallocatechin gallate pergram of dry matter of the cell juice serum; and (vi) between about 0.2and about 1.3 milligrams of (−)-epicatechin gallate per gram of drymatter of the cell juice serum. More particularly, the cell juice serumcan have a catechin content profile as follows: (i) between about 3.0and about 3.5 milligrams of (−)-epigallocatechin per gram of dry matterof the cell juice serum; (ii) between about 5.0 and about 7.0 milligramsof (+)-catechin per gram of dry matter of the cell juice serum; (iii)between about 0.7 and about 1.5 milligrams of (−)-epicatechin per gramof dry matter of the cell juice serum; (iv) between about 1.7 and about2.7 milligrams of (−)-epigallocatechin gallate per gram of dry matter ofthe cell juice serum; (v) between about 0.03 and about 0.07 milligramsof (−)-gallocatechin gallate per gram of dry matter of the cell juiceserum; and (vi) between about 0.5 and about 1.0 milligrams of(−)-epicatechin gallate per gram of dry matter of the cell juice serum.

In one embodiment, fresh biomass of Theacea plants can be used toisolate the bioactive compositions of the present invention. The freshbiomass can be taken from Theacea plants that are of the Camellia and/orEurya genera. Suitable species of the Camellia genus for use in thepresent invention can include, without limitation, Camellia sinensis,Camellia japonica, Camellia reticulate, and Camellia sasanqua. Suitablespecies of the Eurya genus for use in the present invention can include,without limitation, Eurya sandwicensis.

The bioactive composition of the present invention can further include astabilizing agent. Suitable stabilizing agents are those that arecommonly used in the art. Particular suitable stabilizing agents caninclude, without limitation, an emulsifier, a preservative, ananti-oxidant, a polymer matrix, and/or mixtures thereof.

In one aspect of the present invention, the bioactive fraction can havemodulatory activity on at least one mammal cell function. Suchmodulatory activity can include, for example, cell growth inhibitionactivity, cell growth stimulation activity, enzyme secretion activity,enzyme inhibition activity, anti-oxidant activity, UV-protectionactivity, anti-inflammatory activity, wound healing activity, and/orcombinations of these activities. With respect to cell growth inhibitionactivity, such activity can involve growth inhibition of cancer cells.Suitable cancer cells that can be inhibited to grow by the bioactivefractions of the present invention can include, without limitation,breast cancer cells and/or colon cancer cells. The described cell growthinhibition activity can also include growth inhibition of leukemiacells. Suitable leukemia cells that can be inhibited to grow by thebioactive fractions of the present invention can include, withoutlimitation, monocytic leukemia cells.

In another embodiment, the bioactive composition can be effective ininhibiting unwanted hyper-proliferation or hypo-proliferation of skincells and/or inhibiting unwanted uncoordinated enzyme activities orenzyme secretion processes in the skin cells.

In another embodiment, the bioactive composition of the presentinvention can further include a delivery system for systemic or topicaladministration that are commonly used in the art.

The present invention also relates to a bioactive topical formulationsuitable for topical application to a mammal. In one embodiment, thebioactive topical formulation includes a topically effective amount ofthe bioactive composition of the present invention. The bioactivetopical formulation can further include a topically acceptable carrier.Suitable topically acceptable carriers can include, without limitation,a hydrophilic cream base, a hydrophilic lotion base, a hydrophilicsurfactant base, a hydrophilic gel base, a hydrophilic solution base, ahydrophobic cream base, a hydrophobic lotion base, a hydrophobicsurfactant base, a hydrophobic gel base, and/or a hydrophobic solutionbase. In one embodiment, the bioactive composition can be present in anamount ranging from between about 0.001 percent and about 90 percent ofthe total weight of the bioactive topical formulation.

The present invention also relates to a method for inhibitinginflammatory activity in skin tissue of a mammal. This method involvesproviding the bioactive composition according to the present invention.The method further involves applying the bioactive composition to theskin tissue in an amount effective to inhibit inflammatory activity inthe skin tissue. In one embodiment of this method, the bioactivecomposition can further include a stabilizing agent (suitable examplesof which are as described herein). In another embodiment of this method,the bioactive composition can further include a topically acceptablecarrier (suitable examples of which are as described herein).

The present invention also relates to a method of protecting skin tissueof a mammal from ultraviolet light-induced damage. This method involvesproviding the bioactive composition of the present invention. The methodfurther involves applying the bioactive composition to the skin tissuein an amount effective to reduce ultraviolet light-induced damage of theskin tissue and to prevent oxidative damage of the skin tissue. In oneembodiment, the method is useful in protecting skin tissue fromultraviolet light-induced damage caused by ultraviolet light in a rangeof between about 320 and about 400 nanometers. In another embodiment ofthis method, the bioactive composition can further include a stabilizingagent (suitable examples of which are as described herein). In anotherembodiment of this method, the bioactive composition can further includea topically acceptable carrier (suitable examples of which are asdescribed herein).

The present invention also relates to a method for normalizing skindisorders in skin tissue of a mammal. This method involves providing thebioactive composition of the present invention. The method furtherinvolves applying the bioactive composition to the skin tissue in anamount effective to normalize a cell disorder in the skin tissue. In oneembodiment of this method, the bioactive composition can further includea stabilizing agent (suitable examples of which are as describedherein). In another embodiment of this method, the bioactive compositioncan further include a topically acceptable carrier (suitable examples ofwhich are as described herein).

The present invention also relates to a method for isolating a bioactivefraction derived from cell juice of a Theacea plant. This methodinvolves providing a Theacea plant (e.g., in the form of fresh biomass).Suitable Theacea plants for use in this method are as described herein,supra. The Theacea plant (e.g., fresh biomass) is then separated intocell juice and a cell walls component. The cell juice is then treatedunder conditions effective to yield a bioactive fraction. Suitablebioactive fractions include, without limitation, a membrane fraction, amembrane fraction extract, a cytoplasm fraction, a cytoplasm fractionextract, and/or a cell juice serum. The bioactive fraction is thenisolated from the treated cell juice. In one embodiment, the varioussuitable bioactive fractions produced by this method are as describedherein. The present invention further relates to an isolated bioactivecomposition produced by this method.

The present invention also relates to a method for isolating a bioactivefraction derived from a cell walls component of a Theacea plant. Thismethod involves providing a Theacea plant (e.g., in the form of freshbiomass). The Theacea plant (e.g., fresh biomass) is then separated intocell juice and a cell walls component. The cell walls component istreated under conditions effective to yield a bioactive fraction. Thebioactive fraction is then isolated from the treated cell wallscomponent. In one embodiment, the various suitable bioactive fractionsproduced by this method are as described herein. The present inventionfurther relates to an isolated bioactive fraction produced by thismethod.

By way of example, the overall process for preparing the bioactivefractions of the present invention (as described herein above) isschematically shown in FIG. 1. Details of the processing steps arefurther described in the Examples (infra). As depicted in FIG. 1, freshbiomass 10 (e.g., fresh plant biomass) of Theacea plants is subjected togrinding, maceration, and pressing 20 under conditions effective todestroy rigid cell walls, and thereby to yield plant cell juice 30 andcell walls 32. The fresh biomass 10 is also used for conventional teaprocessing 22 to produce positive control 150 for comparative testingand evaluation. Cell juice 30 is subjected to coagulation 40 (e.g.,microwave treatment) to achieve quantitative coagulation of membranefraction components of fresh plant biomass 10. Coagulation 40 issufficient to enable subsequent separation of the coagulated membranefraction from other non-coagulated components of cell juice 30. As shownin FIG. 1, one embodiment of such separation is achieved by cooling andcentrifugation 42 to yield membrane fraction (precipitate) 50 andsupernatant 60, which is free from specific chloroplast membranecomponents such as chlorophyll and phospholipids.

To produce the cell walls fraction extract (i.e., Composition A 110),cell walls 32 are subjected to drying 34 (e.g., several subsequentmicrowave treatments) and then mixing the dried material with water 36under conditions commonly used to prepare conventional teas (e.g.,mixing in water at 85° C.).

To produce the membrane fraction extract (i.e., Composition B 120),membrane fraction 50 is subjected to mixing with solvent 52 and thencentrifugation 54 to yield supernatant 56 and Composition B 120.

To produce the cytoplasm fraction extract (i.e., Composition C 130),supernatant 60 is subjected to coagulation 62 (e.g., isoelectricprecipitation) and centrifugation 64 to yield cytoplasm fraction(precipitate) 70 containing most of the soluble cytoplasm proteins.Cytoplasm fraction (precipitate) 70 is then subjected to mixing withsolvent 72, followed by centrifugation 74 to yield supernatant 76 andthen Composition C 130.

To produce cell juice serum (i.e., Composition D 140), supernatant 60 issubjected coagulation 62 (e.g., isoelectric precipitation) andcentrifugation 64 to yield cell juice serum (supernatant) 66 and thenComposition D 140.

Conventional tea processing 22 of fresh biomass 10 is used to produce,for example, positive control 150 (of various teas, including, forexample, white, green, oolong, and black teas).

Composition A 110, Composition B 120, Composition C 130, Composition D140, and positive control 150 can then be used for filtration and tests80.

The present invention also relates to a device for selectivelydispersing into a liquid low molecular weight and reduced, non-oxidizedcomponents of a bioactive composition. In one embodiment, the deviceincludes a bioactive composition of the present invention. The bioactivecomposition can be enclosed in a filtering pouch. A suitable filteringpouch can be one that is effective in selectively dispersing into aliquid the low molecular weight and reduced, non-oxidized components ofthe bioactive composition. In one embodiment, the pouch includes aselective membrane that allows dispersal of the low molecular weight andreduced, non-oxidized components of bioactive compositions from withinthe pouch into the liquid, but where the membrane inhibits dispersal ofhigh molecular weight and oxidized components from within the pouch intothe liquid. As used herein, the term “low molecular weight and reduced,non-oxidized components” include components of the bioactive compositionof the present invention that are less than or equal to about 5,000Daltons. In one embodiment of this method, the bioactive composition canfurther include a stabilizing agent (suitable examples of which are asdescribed herein). In another embodiment of this method, the bioactivecomposition can further include a topically acceptable carrier (suitableexamples of which are as described herein).

The present invention also relates to a method of making a therapeuticbeverage containing low molecular weight and reduced, non-oxidizedbioactive compositions. This method involves providing a device producedaccording to the method of the present invention. The device iscontacted with a liquid under conditions effective to cause the lowmolecular weight and reduced, non-oxidized components of the bioactivecompositions to disperse into the liquid. In one embodiment of thismethod, the bioactive composition can further include a stabilizingagent (suitable examples of which are as described herein). In anotherembodiment of this method, the bioactive composition can further includea topically acceptable carrier (suitable examples of which are asdescribed herein). A suitable liquid for use in this method can include,without limitation, water. The water can be hot or cold. The presentinvention further relates to a therapeutic beverage produced accordingto this method.

EXAMPLES Example 1 Preparation of Bioactive Compositions Derived fromCamellia sinensis Plants

A schematic of one embodiment of the method of preparing the bioactivecompositions of the present invention is shown in FIG. 1. Below is adescription of relevant aspects of one embodiment of the method of thepresent invention.

Biomass Preparation. Sufficient amounts of fresh Camellia (Camelliasinensis) plant biomass (only top tender young leaf tissue with buds)were harvested to yield approximately 100 kg of dry matter. The level ofdry matter in the fresh biomass was calculated to be 21.70%, requiringharvesting of approximately 461 kg of fresh plant biomass to yield 100kg of dry matter. Care was taken to preserve the inherent moisturecontent of the plant biomass and to avoid wilting due to moisture loss.The harvesting was conducted in such a manner as to avoid or minimizechopping, mashing, and crushing of the collected biomass to avoid thedisruption of the leaf cell structure, which triggers the endogenousenzymatic reactions catalized by phenol-oxidase and peroxidase. Becausethese reactions are intensified with the time of oxidation, all stepswere completed in the shortest possible period of time. For example, theharvested biomass was delivered for processing not more than 10 minutesafter cutting. This was done to minimize exposure of the plant biomassto sun, high temperature, and other negative environmental factors. Awashing step was performed to remove soil particles and other debrisfrom the plants prior to further processing. This washing wasaccomplished by washing the harvested plants for ≦5 minutes in ≦1 kg/cm²water pressure. The residual water wash did not contain any green orbrown pigments, indicating proper water pressure and washing duration.The excess water was removed from the washed plant biomass.

Grinding, Maceration, and Pressing of Plant Biomass. After harvesting,collecting, and washing the plant biomass, the plants then underwentgrinding, maceration, and pressing to extract the intracellular content(i.e., the plant cell juice) and to separate it from the fiber-enrichedcell walls fraction (cell walls fraction). A hammer mill (Model VS 35,Vincent Corporation, FL) having 10 HP engine and set of screens was usedto grind the biomass to yield plant tissue particles of suitably smallsize in a shortest amount of time and without significant increase ofbiomass temperature. The hammer mill was set to produce the maximum sizeof macerated plant particles of ≦0.5 centimeters during ≦10 seconds oftreatment. The biomass temperature was increased only ≦5° C. Ahorizontal continuous screw press (Compact Press “CP-6”, VincentCorporation, FL) was immediately used to extract the plant cell juicefrom the plant. The pressure on the cone of the screw press wasmaintained at a level of 24 kg/cm², with a screw speed of 12 rpm andonly a temperature increase of ≦5° C. This treatment yielded the 185 kgof cell walls fraction having dry matter level 41.39% and 276 kg ofplant cell juice having dry mater level 8.49%.

Preparation of Cell Walls Fraction Extract (Composition A). The aliquotof cell walls fraction having initial dry matter level 41.39% was driedin microwave hood combination (Model GH9115XE, Whirlpool) during 30 secand then cooled during 30 sec. This treatment was repeated several timestill dry matter level in cell walls fraction reached 96.52%. The 66.01of deionized water having temperature 85° C. were added to 4.0 kg of drycell walls fraction and steer with high agitation for 5 min. Theseconditions are in agreement with tea preparation procedure, which isdescribed in D'Amelio, F. S., Botanicals. A Phytocosmetic DeskReference, Boca Raton, London, New York, Washington, D.C.: CRC Press, p.361 (1999), which is hereby incorporated by reference in its entirety(see also the discussions at www.leaftea.com; www.divinitea.com;www.equatorcoffee.com, which are hereby incorporated herein in theirentirety). The mixture was filtered through 4-layers of nylon fabric andthen through the filter having 0.8 μm porous. The pH of obtained cellwalls extract was equal 5.24 and dry matter level was equal 0.84%. Thisextract was further used for tests of its activities.

Separation of the Membrane Fraction from the Cell Juice. The initialplant cell juice having dry matter level 8.49% contained small fiberparticles, which were removed by filtration through four layers of nylonfabric or by using low-speed centrifugation biomass. The filtered plantcell juice was exposed to microwave treatment using a temperature probecontrol. This treatment continued until the temperature of the celljuice reached 60° C. Once coagulation was induced, the treated celljuice was immediately cooled to 40° C. Separation of the membranefraction from the coagulated cell juice was achieved usingcentrifugation at greater than or equal to 3,000 g for greater than orequal to 20 minutes. This yielded a membrane fraction (precipitate) anda cell juice supernatant, which contained a cytoplasm fraction and acell serum fraction (i.e., low molecular weight soluble components). Themembrane fraction having dry mater level 32.89% was used in preparingthe extract of membrane-derived bioactive composition. The cell juicesupernatant was used for further processing to yield cytoplasm fractionand cell juice serum.

Preparation of the Membrane Fraction Extract (Composition B). One partof membrane fraction (10.0 kg) and two parts of Dimethyl Sulfoxide—(20.0kg) were mixed at room temperature for 1 hour with permanent stirring.Then material was centrifuged at greater than or equal to 4,000 g forgreater than or equal to 45 minutes. The precipitate was discarded andsupernatant was filtered through the filter having 0.8 μm porous. Thisfiltrate having dry meter level 6.83%—membrane fraction extract(composition B) was used for further tests of its activities.

Separation of the Cytoplasm Fraction from the Cell Juice Supernatant. Inorder to separate out the cytoplasm fraction, the cell juice supernatantwas subjected to isoelectric precipitation. Precipitation of thecytoplasm fraction was induced using a titration method utilizing 5.0 NHydrochloric Acid (HCl) to bring the pH of the cell juice supernatant to4.0. The separation of precipitated cytoplasm fraction having dry matterlevel 14.5% from supernatant was achieved by centrifugation at greaterthan or equal to 3,000 g for greater than or equal to 20 minutes.

Preparation of the Extract of Cytoplasm Fraction (Composition C). Onepart of cytoplasm fraction (10.0 kg) and two parts of DimethylSulfoxide—(20.0 kg) were mixed at room temperature for 1 hour withpermanent stirring. Then material was centrifuged at greater than orequal to 4,000 g for greater than or equal to 45 minutes. Theprecipitate was discarded and supernatant was filtered through thefilter having 0.8 μm porous. This filtrate having dry meter level3.50%—extract of cytoplasm fraction (composition C) can be used forfurther tests of its activities.

Preparation of Cell Juice Serum (Composition D). After separation ofcytoplasm fraction the supernatant contained suspended particles. Inorder to separate out these particles, the supernatant was centrifugedat greater than or equal to 7,500 g for greater than or equal to 30minutes. The transparent supernatant—cell juice serum was filteredthrough the filter having 0.8 μm porous. This filtrate (composition D)having dry matter level 5.69% was used for further tests of itsactivities.

Preparation of Conventional Tea Extracts—Controls. The same lot of freshCamellia leaves, which was used to preparation of compositions A, B, Cand D was used to produce conventional white and black tea.

The following procedure was used to produce white tea. The fresh biomasscontained 21.70% dry matter was placed for 20 sec in boiling water toinactivate endogenous enzymes—phenol-oxidase and peroxidase. During thisprocedure the leaves were kept in the nylon screen bag. Then treatedleaves were dried in microwave during 30 sec and then cooled during 30sec. This treatment was repeated several times until dry matter level inbiomass reached 93.74%. Then 66.01 of deionized water having temperature85° C. were added to 4.0 kg of dry leaves and steer with high agitationfor 5 min. These conditions are in agreement with tea preparationprocedure, which is described in D'Amelio, F. S., Botanicals. APhytocosmetic Desk Reference, Boca Raton, London, New York, Washington,D.C.: CRC Press, p. 361(1999), which is hereby incorporated by referencein its entirety (see also the discussions at www.leaftea.com;www.divinitea.com; and www.equatorcoffee.com, which are herebyincorporated by reference in their entirety). The mixture was filteredthrough 4-layers of nylon fabric and filtered through the filter having0.8 μm porous. The pH of obtained cell walls fraction extract was equal5.52 and dry matter level was equal 1.10%. This extract was further usedfor tests of its activities.

The following procedure was used to produce black tea. The fresh biomasscontained 21.70% dry matter was kept at 25° C. with periodical (1 hour“on” and 1 hour “off”) aeration until dry matter level reached 35%. Thenleaves were ground (crushed) to the particles having size 2-3 mm. Thisprocedure leads to increase of biomass temperature to approximately 30°C. The ground biomass was placed in the form of layer (2″ high) onplastic conveyor belt for fermentation (oxidation) during 90 min at 25°C. The fermented biomass, which acquired the brown color was dried at130° C. for 30 min to reach the dry matter level 97.5%. Then 66.01 ofdeionized water having temperature 85° C. were added to 4.0 kg of dryleaves and steer with high agitation for 5 min. These conditions are inagreement with tea preparation procedure which is described in D'Amelio,F. S., Botanicals. A Phytocosmetic Desk Reference, Boca Raton, London,New York, Washington, D.C.: CRC Press, p. 361 (1999), which is herebyincorporated by reference in its entirety (see the discussions atwww.leaftea.com; www.divinitea.com; www.equatorcoffee.com, which arehereby incorporated by reference in their entirety). The mixture wasfiltered through 4-layers of nylon fabric and filtered through thefilter having 0.8 μm porous. The pH of obtained cell walls fractionextract was equal 4.96 and dry matter level was equal 1.38%. Thisextract was further used for tests of its activities.

Example 2 Distribution of Dry Matter Regarding Preparation of BioactiveCompositions from Camellia sinensis, Camellia japonica, Camelliareticulate, Camellia sasanqua, and Eurya sandwicensis

Various fractions collected during the production of bioactivecompositions were analyzed and compared for dry matter distribution.Table 1 shows the distribution of 100 kg dry mater among products offractionation of tea plants. It was determined that the process of thepresent invention permits extracted yield conversion into plant celljuices in the range of from about 20 to 30% of initial biomass drymatter. The yield of membrane fractions' dry matter was in the rangefrom 5% to 10% of initial biomass dry matter and from 25% to 35% of celljuice dry matter. Table 1 shows that the yields of cytoplasm fractionsdry matter did not exceed 1.0% of initial biomasses dry matter andsubsequently 2.5% of cell juice supernatant dry matter. Most of celljuice supernatant dry matter was concentrated in cell juice serum. Thecell walls fraction, membrane fraction and cytoplasm fraction were usedas the sources for preparation of their extracts, which are categorizedas bioactive compositions. The cell juice serum was directly used “asis” as subsequent bioactive composition having no exogenous solvents.

TABLE 1 Distribution of 100 kg Dry Matter Among Products ofFractionation of Fresh Biomass Plant Source Product Camellia sinensisInitial Biomass 100.0 Cell Walls Fraction 76.6 Cell Juice 23.4 MembraneFraction 6.5 Cytoplasm Fraction 0.6 Cell Juice Serum 16.3

It should be noted that the three selected materials are the mostdiversified representation of all functional structures, which exists infresh plant tissue. Only soluble cell juice serum has physico-chemicalproperties, which allows the direct administration to the commonly usedin vitro testing systems. The cell walls fraction, membrane fraction andcytoplasm fraction were used as raw materials for extraction withsolvents. Because cell walls fraction is structurally similar toconventional tea plant products, this fraction was extracted with waterto provide the best comparison with conventional teas. The membranefraction was extracted with Dimethyl Sulfoxide, which facilitates theeffective solubilization of both hydrophobic and hydrophilic componentsintegrated in chloroplast and mitochondria structures. The cytoplasmfraction was extracted with water. The cell juice serum was used “asis.”

Table 2 shows the yield of all four tested bioactive compositions: cellwalls fraction extract (composition A), membrane fraction extracts(composition B), extract of cytoplasm fraction (composition C), celljuice serum (composition D) and controls—white tea extract or black teaextract from 100 kg of initial biomass dry mater.

TABLE 2 Yield of Bioactive Compositions from 100 kg of Initial BiomassPlant Source Product Camellia sinensis Initial Biomass 100.0 CompositionA (Cell Walls Fraction Extract) 14.35 Composition B (Membrane FractionExtract) 2.37 Composition C (Cytoplasm Fraction Extract) 0.2 CompositionD (Cell Juice Serum) 16.3 Control (Extract of White Tea or Black Tea)15.14 . . . 19.36

Table 2 shows that the total yield of bioactive compositions A, B, C andD from 100 kg of dry matter of Camellia sinensis equals 33.3%, whichvery significantly exceeds the yield of conventional tea process—15.14 .. . 19.36%.

Example 3 Comparison of Composition A (Cell Walls Fraction Extract) andConventional Tea Extracts

Various parameters of bioactive composition A and conventional white andblack tea extracts obtained from the same batch of fresh Camelliasinensis were measured and subsequent results of these are presented inTable 3 (The used experimental methods are described in Examples 9 and20, and in the U.S. Patent Application Publication No. 2003/0175235,which is hereby incorporated by reference in its entirety).

TABLE 3 Various Parameters of Bioactive Composition A and Extracts ofWhite and Black Teas. Extract of Cell Walls Controls Fraction Extract ofExtract of Parameter (Composition A) White Tea Black Tea Dry Matter, %0.84 1.10 1.38 pH 5.24 5.52 4.96 Conductivity, mS/cm 1.57 2.23 5.32Total Dissolved Solids, g/L 0.78 1.23 2.71 Redox Potential, mV 123 159188 Area under the 5.727 6.604 4.616 Spectra Curve (ASC) 200-450 nm, Abs· nm ASC: Dry Matter 6.818 6.004 3.345 Superoxide Scavenging 26.3 114.5227.1 Activity (ICR₅₀), μg DM/ml Color (1 . . . 10 scale) 9.6 4.7 7.5Flavor (1 . . . 10 scale) 9.3 6.1 6.6 Mouthfeel (1 . . . 10 scale) 9.46.5 5.3

Table 3 shows that cell walls fraction extract has lower levels of drymatter, electrolytes and dissolved solids compare to conventional whiteand black tea extracts. The UV/VIS spectral data show that cell wallsfraction extract has the highest specific value of the area under thespectra curve, i.e. this particular Camellia product (composition A) hasthe highest level of optically active constituents per unit of drymatter. Additionally, the cell walls fraction extract has the loweramount of redox potential, which indicates that this composition is lessoxidized then conventional white and black tea extracts. The cell wallsfraction extract demonstrated superoxide scavenging activity, resultingin 50% inhibition of cytochrome c reduction (ICR₅₀) at a much lowerconcentration then white and black tea extracts. A QualitativeDescriptive Analysis (“QDA”) test method was selected to systematicallycharacterize and quantify teas based on color, flavor, and mouthfeel,which govern acceptability of tea beverages. The QDA method employs atrained panel of expert tasters to quantify the above attributes of teabeverages relative to defined reference standards. The comparativeevaluation of the color, flavor and mouthfeel of teas demonstrated thatcell walls fraction extract significantly exceed the samecharacteristics of conventional teas.

Thus, cell walls fraction, which was obtained from fresh Camelliabiomass without any fermentation (oxidation) and heat treatment, isdistinct from all other teas (Wilson et al., eds., Tea: Cultivation toConsumption, London: Chapman Hall (1992), which is hereby incorporatedby reference in its entirety). Additionally, the key Camellia enzymes(phenol-oxidase and peroxidase) always remain within conventional teas.Instead, the present invention includes separation of fresh Camellialeaves to cell walls fraction and cell juice, which is enriched by theseenzymes and thus cell walls fraction does not contain endogenousphenol-oxidase and peroxidase. Therefore, cell walls fraction must becategorized as a new tea category having fundamental differencescompared with white, green, oolong, and black teas. This novel cellwalls fraction tea can be used in either loose or bag form or othermanifestations to prepare a broad spectrum of drinks, beverages, andadditives to nutriceutical and functional food products.

Example 4 Preparation of Bioactive Compositions for DifferentApplications

All bioactive compositions can be used as solutions, suspensions,dispersions, pastes or dried powders incorporated into a variety offormulations for systemic or topical administrations. The solubilizedforms of compositions can be filtrated through filters having the 0.2 μmporous to completely remove non-completely solubilized small particlesand endogenous microorganisms. The dry matter level in bioactivecompositions before and after sterilized filtration is presented inTable 4.

TABLE 4 Level of Dry Matter in Bioactive Compositions Before (numerator)and After (denominator) Sterilized Filtration Plant Source ProductCamellia sinensis Composition A 0.84 (Extract of Cell Walls Fraction)0.72 Composition B 6.83 (Extract of Membrane Fraction) 6.12 CompositionD 5.69 (Cell Juice Serum) 5.59 Control 1.10 (Extract of White Tea) 1.03

Table 4 shows that dry matter levels in all bioactive compositions weredecreased after sterilizing filtration. However this decrease did notlead to any loss or significant reduction of their biological activitiesand was in the range 2-14%. Therefore major part of compositions ispresented by soluble bioactive ingredients.

Fresh Camellia leaves contain relatively low molecular weight (reduced,non-oxidized) ingredients. As a result of oxidation and polymerizationprocesses in the manufacturing of conventional teas the above potentingredients are transformed into the parts of high molecular weightsubstances having relatively low activity.

The bioactive compositions of the present invention are obtained withoutfermentation (oxidation) and excessive heat treatment. This in turnprevents the irreversible loses of fresh plant activities, which can bedelivered with maximum potency using, for example, novel tea bag oranalogous delivery systems. Instead of conventional paper tea bags,which allow all soluble tea ingredients to move through large pores tothe surrounding water, the novel tea bag is made from semi-permeablemembrane. This bag contains bioactive composition inside and allows onlyingredients having molecular weight below certain membrane cut off (forexample, 5,000 Dalton) to penetrate to the surrounding water phase. Theingredients having higher molecular weight remain inside the bag andthus are not included in the beverage.

Therefore, the cut off of ingredients having molecular weight above acertain level allows the production of a beverage having no oxidizedingredients because all oxidized ingredients of bioactive compositionshaving molecular weight above a certain level remain inside the bag. Thenovel tea bag design can be based on pyramidal tea bag construction,which utilizes dialysis membrane tube. The design of novel tea bag canalso include thin plastic frame having bioactive composition inside andtwo transparent surfaces built from semi-permeable membrane. Theselection of particular membrane cut off value is determined by the typeof bioactive composition, but in general higher cut off enabling releaseinto surrounding water phase of a higher percent of composition's drymatter lower amount of the redox potential is preferable.

Example 5 Preparation of Topical Ingredient SF Derived from Cell JuiceSerum

Cell juice serum (composition D) cannot be used as an active ingredientof topical products due to the lack of stability and deterioration ofcolor and odor. The described procedure allows for the refinement ofcell juice serum fraction to yield a stable and active topicalingredient SF (this procedure is similar to previously described in U.S.Patent Application Publication No. 2003/0175235, which is herebyincorporated by reference in its entirety). The refinement of the celljuice serum involved the following steps: heat treatment, cooling,filtration, and stabilization. Refinement was performed immediatelyafter separation of the cell juice serum from the cytoplasm fraction asdescribed in Example 1. The cell juice serum was exposed to microwavetreatment using a temperature probe control. This treatment continueduntil the temperature of the cell juice serum reached 99° C. (90° C. wasrequired as was previously described in U.S. Patent ApplicationPublication No. 2003/0175235, which is hereby incorporated by referencein its entirety). Once coagulation was induced the treated cell juiceserum was immediately cooled to 10° C. The coagulated cell juice serumwas vacuum filtrated through filter having porous 0.8 μm (double layersof Whatman No. 2 filters were used in U.S. Patent ApplicationPublication No. 2003/0175235, which is hereby incorporated by referencein its entirety). The precipitate was discarded and the resulting celljuice serum filtrate was used for further processing (i.e.,stabilization). Stabilization of the cell juice serum filtrate wasachieved by adding preservatives (no exogenous anti-oxidant was requiredas was previously described in U.S. Patent Application Publication No.2003/0175235, which is hereby incorporated by reference in its entirety)and incubating the mixture until complete solubilization was achieved.The preservatives used included the following: 0.1% potassium sorbate,0.1% sodium benzoate, 0.1% sodium methyl paraben, and 0.1% citric acid.This preparation resulted in the production of 16.3 kg of dry matteryield (or approximately 286 Liters) of the topical ingredient SF, whichwas used for characterization of its physico-chemical and bioactivequalities. The recommended storage conditions for topical ingredient SFinclude storage in a closed container protected from light at atemperature of between 15° C. and 25° C.

Example 6 Product Specifications of Topical Ingredient SF Derived fromCell Juice Serum Fraction

Topical Ingredient SF was prepared according to the process describedabove in Example 5. Analyses of topical ingredient SF were conducted todetermine its various physico-chemical, microbial, cytotoxicity, andbioactivity characteristics, as described below. Topical ingredient SFis a clear liquid, which has a light-yellow-brown color and alight-characteristic odor. No solvent (i.e. glycol, oil, or water) wasadded to the carrier medium. Table 5 summarizes the Physical andChemical data of topical ingredient SF.

TABLE 5 Physical and Chemical Parameters of Topical Ingredient SFParameter Method Results Solid Content, % See Example 20, “Method 1”5.04 Specific Gravity, g/cm³ USP <841> 1.015 Color Gardner Scale 5-6Refractive Index USP <831> 1.312 PH USP <791> 4.0 Redox Potential, mVSee reference [1] 75 Conductivity, S/m See reference [2] 1.02References: [1] Handbook of Chemistry and Physics, 80 ^(th) Edition, CRCPress, 1999-2000, 5-90; [2] Handbook of Chemistry and Physics, 80 ^(th)Edition, CRC Press, 1999-2000, 8-21, which are hereby incorporated byreference in their entirety.

Table 6 describes the UV-Spectra data regarding topical ingredient SF.

TABLE 6 UV-Spectra of Topical Ingredient SF (1:500 Dilution) PeakParameter Method Results #1 Start, nm USP <197> 450 Apex, nm ″ 266.5End, nm ″ 247 Height, Abs ″ 0.231 Area, Abs x nm ″ 13.676 #2 Start, nmUSP <197> 247 Apex, nm ″ 204 End, nm ″ 200 Height, Abs ″ 1.396 Area, Absx nm ″ 32.413

The microbial analysis conducted in accordance with the followingprocedure (USP <61>) demonstrated that topical ingredient SF containsless then 100 colony forming units per gram of sample and has nopathogens (E. coli, Candida albicans, Pseudomonas sp. and Staphylococcusaureus). This data demonstrates that topical ingredient SF satisfies theindustry requirements for ingredients of topical products.

Topical ingredient SF was determined to be stable (i.e., maintainingphysical and chemical integrity) for at least 12-18 months while storedat a temperature of between 15 and 25° C. in a closed containerprotected from light. Topical ingredient SF is a biodegradable product.In a controlled clinical evaluation, topical ingredient demonstrated thebiological activities, which are summarized in Table 7.

TABLE 7 Biological Activities of Topical Ingredient SF Activity Methodμg DM/ml Superoxide Scavenging Activity (ICR₅₀) See Example 20, 69.5“Method 7” Elastase Inhibitory (IC₅₀) See Example 20, 32.3 “Method 5”MMP-9 Inhibitory (IC₅₀) See Example 20, 14.6 “Method 6” TrypsinInhibitory (IC₅₀) See reference [1] 7.8 Reference: [1] Cannel et al.,Planta Medica 54: 10-14 (1988), which is hereby incorporated byreference in its entirety.

Table 7 shown that topical ingredient SF demonstrated superoxidescavenging ability. In a controlled clinical evaluation, topicalingredient SF demonstrated a 50% inhibition of cytochrome c reduction(ICR₅₀) at a concentration 69.5 μg dry matter per ml. The ICR₅₀ ofpositive control (rosmarinic acid)=26.5 μg/ml. In addition toanti-oxidant properties, topical ingredient SF demonstratedantiproteolytic activities against peptide hydrolases, for exampleelastase, gelatinase B or so-called matrix metalloproteinase 9 (MMP-9),and trypsin. Among these enzymes the unique position belongs to elastaseand MMP-9, which act synergistically and play an extremely importantrole in skin inflammation. It should be noted, that both MMP-9 andelastase are secreted by white blood cells (neutrophils) and theseenzymes are the key enzymes in the final pathway leading toinflammation. It is generally agreed that if preparation can inhibitboth enzymes (elastase and MMP-9), such preparation is considered to bevery effective to treat inflammatory processes.

It should be noted that skin aging processes, sunburns, formation ofwounds and scars have the very same inflammation mechanism, whichinvolves both MMP-9 and elastase. Thus, topical ingredient SF capable ofinhibiting both of the above enzymes has very wide spectrum ofapplications, among which are inflammatory injury because the followingreasons:

-   -   a. These two enzymes can synergize to degrade all the components        of extracellular matrix of human tissue;    -   b. Elastase can inactivate the body's own inhibitory defense        against MMP-9; and    -   c. MMP-9 can inactivate the body's own inhibitory defense        against elastase.        The combination of anti-inflammatory and anti-oxidant properties        of topical ingredient SF suggests that this hydrophilic        preparation based on bioactive composition D is capable to act        systemically on very fundamental skin disorder problems.

Example 7 Preparation of Topical Ingredient MF Derived from MembraneFraction

The freshly obtained membrane fraction is a paste having intensive colorand specific odor. This fraction is represented predominantly bychloroplasts and its composition includes predominantly phospholipids,membrane proteins, chlorophyll, and carotenoids. The drying of membranefraction results in irreversible loses of many valuable propertiesrequired for the exploration of membrane fraction as a topicalingredient. Without drying, the unstable membrane fraction is quicklytransformed into the dark color un-dispersible and insolubleconglomerates having a strong and non-characteristic odor. As result,such material cannot be used as a topical ingredient. The proceduredescribed below allows for transformation of freshly obtained membranefractions into stable and active topical ingredients (this procedure issimilar to previously described in the U.S. Patent ApplicationPublication No. 2003/0175235, which is hereby incorporated by referencein its entirety).

Immediately after separation of the membrane fraction from cell juiceaccording to the process described above in Example 1, the membranefraction was stabilized and incorporated into a polymer matrix. Toprepare approximately 100 grams of topical ingredient MF the cellmembrane fraction was stabilized by mixing it with non-ionic emulsifierPolysorbate 80 (Tween 80) and antioxidants (Tenox 4). Specifically, 20grams of fresh membrane fraction was mixed vigorously with 3.5 grams ofTween 80 and 0.1 gram of Tenox 4 (solution of Butylated Hydroxyanisoleand Butylated Hydroxytoluene in oil) until homogeneous, while avoidingaeration during mixing.

Once stabilized, the membrane fraction was incorporated into a polymermatrix (i.e., a dispersion of polymeric emulsifier, acrylates/C10-C30acrylate crosspolymer). The polymer matrix was prepared by dispersing0.9 grams of Pemulen TR-2 in 69.2 grams of warm deionized water andmixing until uniform using moderate agitation, while avoiding aeration.In parallel, 5 grams of Glycerin and 1.0 gram of Phenonip (mixture ofPhenoxyethanol (and) Methylparaben (and) Butylparaben (and) Ethylparaben(and) Propylparaben) were combined in a separate vessel and mixed untiluniform. With moderate agitation, the phases containing Pemulen andGlycerin with Phenonip were combined and mixed until uniform. Toincorporate the membrane fraction into the polymer matrix, the phasecontaining the membrane fraction, Tween 80, and Tenox 4 was added to thephase containing the Pemulen, Glycerin, and Phenonip, and then mixedwith vigorous agitation while avoiding aeration. Stabilization of themembrane fraction mixture was achieved by neutralizing it with 18%aqueous solution of sodium hydroxide (NaOH) and mixed vigorously toproduce a uniform system having a pH of 5.0±0.4. This preparation, whichstarted from 100 kg of fresh Camellia biomass (approximately 461 kg offresh leaves having 21.7% dry matter), resulted in the production of11.85 kg of Dry Matter yield (or approximately 172 liters) of topicalingredient MF, which was used for characterization of itsphysico-chemical and bioactive qualities. The recommended storageconditions for topical ingredient MF include storage in a closedcontainer protected from light at a temperature between 2 and 8° C.

Example 8 Product Specifications of Topical Ingredient MF Derived fromMembrane Fraction

Topical ingredient MF was prepared according to the process describedabove in Example 7. Analyses of topical ingredient MF were conducted todetermine its various physico-chemical, microbial, cytotoxicity, andbioactivity characteristics, as described below. Topical ingredient MFis an opaque gel, which has a green-brown color and light-characteristicodor. Topical ingredient MF was formulated utilizing the natural celljuice constituents gelled with a polymer to assure the highest level ofpurity uniformity, compatibility, stability, safety and efficacy.

Table 8 describes the physical and chemical data of topical ingredientMF.

TABLE 8 Physical and Chemical Parameters of Topical Ingredient MFParameter Method Results Non-Volatile Residue (NVR), % See Example 20,6.9 “Method 2” Specific Gravity, g/cm³ USP <841> 1.035 Viscosity, cpsUSP<911> 18,700 pH USP <791> 4.6 Total Carotenoids, % NVR See Example20, 0.36 “Method 4” Lutein, % NVR See Example 20, 0.34 “Method 4”

Table 9 summarizes the L*a*b* values data regarding topical ingredientMF.

TABLE 9 L*a*b* Values of Topical Ingredient MF Parameter Method ResultsL* See Example 20, “Method 3” 30.27 a* ″ 27.36 b* ″ 42.56

Microbial analyses demonstrated that topical ingredient MF satisfies theindustry requirements for topical ingredients with regard to CFUs andabsence of pathogens (USP <61>).

Topical ingredient MF was determined to be stable (i.e., maintainingphysical and chemical integrity) for at least 12-18 months while storedat a temperature of between 2 and 8° C. in a closed container protectedfrom light. Topical ingredient MF is a biodegradable product. In acontrolled clinical evaluation, topical ingredient MF demonstrateselastase inhibitory activity and trypsin inhibitory activity. Table 10summarizes certain bioactivity results for topical ingredient MF.

TABLE 10 Bioactivity Results of Topical Ingredient MF Activity MethodIC₅₀ (μg/ml) Elastase Inhibitory (IC₅₀) See Example 20, “Method 5” 12.3MMP-9 Inhibitory (IC₅₀) See Example 20, “Method 6” 5.6 TrypsinInhibitory (IC₅₀) See reference [1] 3.8 Reference: [1] Cannel et al.,Planta Medica 54: 10-14 (1988), which is hereby incorporated byreference in its entirety.

Table 10 shown that topical ingredient MF demonstrated propertiessimilar to topical ingredient SF (see Example 6). Although topicalingredient MF has no superoxide scavenging activity, it demonstrateshigher specific enzyme inhibition activities than topical ingredient SF.Thus topical ingredient MF, which is based on bioactive composition B,should be considered as a potent multiphase anti-inflammatory ingredienthaving broad applications for treatment of skin disorders.

Example 9 Spectral Analyses of the Bioactive Compositions Derived fromCamellia sinensis Plants

Introduction to Spectral Analyses. Ultraviolet (UV) radiation hasdamaging effects on human skin. Short-term effects include tanning andsunburn, while the long-term effects of cumulative UV exposure includephotoaging of the skin and increased risk of skin cancer. Ultravioletskin injury is mediated by oxidative damage, and a number of plantextracts with antioxidant activity are showing promise as protectiveagents: grape seed extract (Carini et al., “Protective Effect ofProcyanidines from Vitis vinifera Seeds on UV-Induced Photodamage: Invitro and In vivo Studies,” Proceedings of the 19th IFSCC Congress3:55-63 (1996), which is hereby incorporated by reference in itsentirety), lycopene (Di Mascio et al., “Lycopene as the Most EfficientBiological Carotenoid Singlet Oxygen Quencher,” Archives of Biochemistryand Biophysics 274:532-8 (1989); and Ribaya-Mercado et al., “SkinLycopene is Destroyed Preferentially Over β-Carotene During UltravioletIrradiation in Humans,” Journal of Nutrition 125:1854-9 (1995), whichare hereby incorporated by reference in their entirety), silymarin(Morazzoni et al., “Silybum marianum (Carduus marianus),” Fitoterapia66:3-42 (1995); Katiyar et al., “Protective Effects of Silymarin AgainstPhotocarcinogenesis in a Mouse Skin Model,” Journal of the NationalCancer Institute 89:556-66 (1997), which are hereby incorporated byreference in their entirety), and especially green tea extract which hashigher efficacy compared with extracts produced from other plant sources(Katiyar et al., “Protection Against Ultraviolet-B Radiation-InducedLocal and Systemic Suppression of Contact Hypersensitivity and EdemaResponses in C3H/HeN Mice by Green Tea Polyphenols,” Photochemistry andPhotobiology 62:855-61 (1995); Ruch et al., “Prevention of Cytotoxicityand Inhibition of Intercellular Communication by Antioxidant CatechinsIsolated from Chinese Green Tea,” Carcinogenesis 10:1003-8 (1989); Wanget al., “Protection Against Ultraviolet B Radiation-InducedPhotocarcinogenesis in Hairless Mice by Green Tea Polyphenols,”Carcinogenesis 12:1527-30 (1991), which is hereby incorporated byreference in its entirety).

It was found that the leaves of the tea plant (Camellia sinensis) have ahigh content of polyphenols with antioxidant activity including(−)-epicatechin, (−)-epicatechin-3-gallate, (−)-epigallocatechin, and(−) epigallocatechin-3-gallate. Green tea extract has shown anantioxidant activity against hydrogen peroxide and the superoxideradicals and prevention of oxidative cytotoxicity. The extract can alsoprevent the inhibition of intercellular communication, a possiblemechanism of tumor promotion. There is a close association betweenUV-induced immune suppression and the development of skin cancer, andgreen tea extract has been found to protect against inflammation andimmune suppression caused by UV-B radiation. Green tea extract givenorally in drinking water or applied topically protects againstUV-B-induced skin carcinogenesis in animal models. These resultsindicate that green tea extract taken orally may help to prevent skincancer.

Although the UV protection properties of Camellia products have beenestablished, the greater potential of tea plant as a source foreffective protection of the skin against sun damage has not fullyexplored due to the limitations of conventional technology, which isdriven towards focusing on a limited to relatively narrow band of activeingredients: predominately cathechins

Comparative UV protection properties studies described herein between“novel” and “conventional” Camellia products have now demonstrated thatthe technology of “fresh Camellia fractionation” is capable of yieldingmore potent products. The comparison was made by utilizing the methodscommonly used to determine spectral properties of solutions and in-vitrosun protection factor (SPF).

Methodology A: UV/VIS Spectra. UV/VIS spectra of Camellia products in200-450 nm region were obtained using pharmacopoeia compliantSpectrophotometer Ultrospec 4300 Pro (Amersham Biosciences Ltd.,Buckinghamshire, England). The spectral parameters of diluted indistilled water Camellia products were determined according to theprocedure described in USP <197>.

Methodology B: Absorbance Spectra. Absorbance spectra of Camelliaproducts in 250-450 nm region were obtained using UV-1000S TransmittanceAnalyzer (Labsphere, Inc., North Sutton, N.H.) and Vitro-Skin® testingsubstrate (IMS Testing Group, Milford, Conn.), which mimics the surfaceproperties of human skin. It contains both optimized protein and lipidcomponents and is designed to have topography, pH, critical surfacetension and ionic strength similar to human skin.

The Camellia samples were uniformly spread on a surface of pre-hydratedsubstrate (application dose=2.0 μl/sq. cm). After 15 min afterapplication the initial absorbance spectra were taken via five (5)replications. Then the substrates with applied products were irradiatedby broad-spectrum solar light simulator (Model 16S-300 Single Port,Solar Light Company, Inc., Philadelphia, Pa.) equipped with 300-Wattxenon lamp. The dose control system PMA 2100-DCS allowed precisioncontrol of the dose delivered to a sample.

Immediately after irradiation (irradiation dose=60 Joules/sq. cm)absorbance spectra from the same spot were taken in (5) fivereplications. The absorbance spectra of samples before and afterirradiation were used for statistical analysis.

Samples. The following bioactive compositions, which were preparedaccording to the process described above in Example 1 were evaluated:composition A (cell walls fraction extract having 0.84% dry matter),composition B (membrane fraction extract having 6.83% dry matter),composition D (cell juice serum having 5.69% dry matter). Extract ofconventional white tea having 1.10% dry matter and extract ofconventional black tea having 1.38% dry matter were used as controls.All samples were obtained from the same batch of fresh Camellia, whichwas collected at Charleston Tea Plantation, SC. These samples did notcontain any additives.

Analyses. It was found that all Camellia samples have high UV absorbancevalues and thus they were diluted with distilled water. The UV-VISspectra of diluted Camellia products are presented on FIGS. 2 and 3.

The spectra of all liquid samples have certain similarities. Forexample, positions of peaks are varied in relatively narrow ranges ofλ_(max1)=269-274 nm and λ_(max2)=205-208 nm, which indicate the presenceof aromatic rings and conjugated systems of σ−π bonds in all testedsamples. However, the apex value of peaks, area under each peak andtotal area under integral spectral curves are different (Table 11),which suggest that tested samples have different compositions ofoptically active constituents.

TABLE 11 Parameters of UV/VIS Spectra of Camellia Products Area underPeak # 1 Peak # 2 the Spectra Start, λ_(max1), End, Height, Area Start,λ_(max2), End, Height, Area Curve*, nm nm nm Abs % nm nm nm Abs % Abs ·nm White Tea 450 269 250 0.114 28.1 250 205 200 0.816 71.9 6.604 ExtractBlack Tea 450 272 250 0.094 37.3 250 205 200 0.452 62.7 4.616 ExtractCell Walls 450 272 248 0.134 35.8 248 205 200 0.614 64.2 5.727 FractionExtract Membrane 450 274 251 0.944 16.2 251 208 200 11.98 83.8 80.133Fraction Extract Cell Juice 450 271 249 0.692 26.3 249 205 200 5.22873.7 39.599 Serum *The Area under Spectra values were normalized basedon dilution of the samples.

The comparison of areas under integral spectral curves obtained from 200nm to 450 nm clearly demonstrates that membrane fraction extract(composition B) and cell juice serum (composition D) had the higherabsorption values (Table 11). The ratio “Area under Spectra:Dry Matter”indicates that specific absorption value of the samples is increasing inthe following sequence: black tea extract>white tea extract>cell wallsfraction extract>cell juice serum>membrane fraction extract (Table 12).

TABLE 12 Selected Spectral Characteristics of Camellia Products Dry Areaunder Ratio: Area under Ratio: Matter Spectra, Area under Spectra^((200-450 nm)) Spectra^((290-400 nm)) Area under Spectra^((290-400 nm)) % Abs · nm Dry Matter Abs · nm Dry Matter White TeaExtract 1.10 6.604 6.004 0.812 0.738 Black Tea Extract 1.38 4.616 3.3450.854 0.619 Cell Walls Fraction 0.84 5.727 6.818 0.776 0.924 ExtractMembrane Fraction 6.83 80.133 11.733 4.304 0.630 Extract Cell JuiceSerum 5.69 39.599 6.959 4.320 0.759

Based on the comparison of absorption values, novel bioactivecompositions appear to be more effective protectors of the skin againstsun damage than extracts of conventional white tea and black tea. Itshould be pointed out, that UV protection properties of Camelliaproducts should be better estimated using absorption data related to thearea from 290 nm to 400 nm because this particular part of spectra isresponsible for UV induced damage of the skin (Sayre et al., “A Methodfor the Determination of UVA Protection for Normal Skin,” Journal ofAmerican Academy of Dermatology 23: 429-40 (1990), which is herebyincorporated by reference in its entirety). Although absorption oftested liquid samples in the area 290-400 nm contributes only ˜10% oftotal UV/VIS absorption, novel Camellia compositions have higherabsorption in the above spectral area as well.

Thus the data related to the diluted solutions of Camellia samplesprovided initial estimation of UV protection potency of tested products,which was further evaluated using the Vitro-Skin® testing substrate (IMSTesting Group, Milford, Conn.). The results are presented in FIGS. 4-13.It was found that novel Camellia compositions and extracts ofconventional white tea and black tea have different spectralcharacteristics even after they were applied on substrate inconcentrations, which were equalized with respect to dry matter level(FIGS. 4 and 5).

The spectral of Camellia samples, which were applied on Vitro-Skin®testing substrate (IMS Testing Group, Milford, Conn.) included from fourto two characteristics peaks, which have different apex values (Table13).

TABLE 13 Parameters of Absorbance Spectra of Camellia Products Appliedon Vitro-Skin ® Testing Substrate Peak # 1 Peak # 2 Peak # 3 Peak # 4Height, Height, Height, Height, λ_(max4) Abs λ_(max3) Abs λ_(max2) Absλ_(max1) Abs White Tea Extract 260 0.389 286 0.461 330 0.163 392 0.076Black Tea Extract 260 0.399 286 0.373 330 0.175 390 0.105 Cell WallFraction 260 0.555 286 0.569 330 0.203 389 0.118 Extract MembraneFraction 260 0.394 286 0.549 — — 394 0.059 Extract Cell Juice Serum 2600.431 286 0.517 — — — —

It should be pointed out, that parameters of characteristic peaks ofCamellia products in solutions (Table 11) were very different comparedwith characteristic of Camellia products, which were applied onVitro-Skin® testing substrate (IMS Testing Group, Milford, Conn.) (Table13). As control experiments show, the lower pH level (˜5.5) onVitro-Sin® surface can not be responsible for the above differences,which probably are the results of chemical interactions between Camelliaproducts and ingredients used for preparation of Vitro-Skin® testingsubstrate (IMS Testing Group, Milford, Conn.) substrate. Thus,Vitro-Skin® testing substrate (IMS Testing Group, Milford, Conn.)contains both protein and lipid components which could chemicallyinteract with Camellia phenolic constituents. It should be noted, thatthe shifts in spectral properties of tested products were observed forall bioactive compositions as well as for extracts of conventional teas.

Spectra of novel Camellia compositions and extracts of conventionalCamellia products “as is” were also compared, taking into account thedifferent dry matter contents of tested samples. The absorbance spectraof Camellia products applied on Vitro-Skin® testing substrate (IMSTesting Group, Milford, Conn.) in equal volumes are presented on FIG. 6.

Although there are some similarities between spectra of cell wallsfraction extract and white tea extract, the first product has higherabsorbance in the area 250-280 nm and in near UV area. It should benoted that higher absorbance does not correspond with dry matter level,which is higher in white tea extract (1.10%) compared with cell wallsfraction extract (0.84%). It suggests that the compositions of these twosamples are not identical and that cell walls fraction extract also haslower conductivity consisting of greater non-dissociated opticallyactive ingredients responsible for high absorbance (see data presentedin Table 3).

Additionally it was found that the spectra of membrane fraction extract(composition B) and cell juice serum (composition D) are different fromspectra of cell walls fraction extract (composition A) and white teaextract. Thus, membrane fraction extract and cell juice serum spectraagain indicate that these products have different composition than whitetea extract and cell walls fraction extract. At the same time, thespectral data suggest that the compositions of membrane fraction extractand cell juice serum are not identical. For example, membrane fractionextract has three characteristic peaks at 260 nm, 286 nm and 394 nm. Thecell juice serum spectra contain two peaks at 260 nm and 286 nm.

Comparison of these two spectra indicates that membrane fraction extracthas approximately two times higher extinction then cell juice serum,although difference in dry matter levels is only ˜1%. Thus, four testedCamellia products have significantly different compositions ofconstituents, which are optically active in the region 250-450 nm.

The data related to the quantitative comparison of Camellia products arepresented in Table 14.

TABLE 14 Selected Characteristics of Camellia Products Applied ofVitro-Skin ® Testing Substrate Area under Area under Area under Spectra^((290-400 nm)) Spectra^((250-450 nm)) Spectra^((290-400 nm)) Area underSpectra^((250-450 nm)) Abs · nm Abs · nm % White Tea Extract 15.5779.312 59.78 Cell Walls Fraction Extract 18.571 10.422 56.12 MembraneFraction Extract 89.148 53.803 60.35 Cell Juice Serum 60.861 35.16157.77

Table 14 shows that absorption of the samples in the area 250-450 nm andin the area 290-400 nm is increasing in the following order: white teaextract>cell walls fraction extract>cell juice serum>membrane fractionextract. This sequence is completely in agreement with the sequence ofspecific absorption values of Camellia products tested in the dilutedsolutions (Table 12). The contribution of the absorbance in the 250-400nm region to the absorbance of the spectra taken from 250 nm to 450 nmreached ˜55-60% when tested samples were applied on Vitro-Skin® testingsubstrate (IMS Testing Group, Milford, Conn.).

It should be noted that the spectra of novel Camellia compositions indiluted solutions and after their application on Vitro-Skin® testingsubstrate (IMS Testing Group, Milford, Conn.) substrate are remarkablydifferent. These differences are both quantitative and qualitative formembrane fraction extract and cell juice serum. Thus in the range from250 nm to 450 nm membrane fraction extract in solution peaks at 274 nm.The same membrane fraction extract when applied on Vitro-Skin® testingsubstrate (IMS Testing Group, Milford, Conn.), did not show anycharacteristic peak at the above wavelength, but instead had twocharacteristic peaks at 260 nm and 286 nm (FIG. 7A).

The similar pattern in spectral properties was observed for cell juiceserum, although additional absorption at ˜360 nm was identified in thesample applied on Vitro-Skin® testing substrate (IMS Testing Group,Milford, Conn.), but such phenomena was not registered in UV/VIS spectraof the same composition in the solution (FIG. 7B).

It should be pointed out that above differences between spectra may bethe results of chemical interaction between novel Camellia compositionsand surface of Vitro-Skin® testing substrate (IMS Testing Group,Milford, Conn.) having protein and lipid components, which mimic humanskin. These interactions led to the drastic increase of the absorbancein the spectral region responsible for damaging effect of UVirradiation, i.e., novel Camellia compositions have significant UVprotection potencies. Control experiments with Barley (Hordeum vulgare)(FIG. 8A) and Sage (Salvia officinalis) (FIG. 8B) cell juice serumsshown that the differences in the spectra of the same samples insolution and after their application on Vitro-Skin® testing substrate(IMS Testing Group, Milford, Conn.) were not observed for plant sourcesother than Camellia. Thus, the spectral shift in Camellia products afterapplication on Vitro-Skin® testing substrate (IMS Testing Group,Milford, Conn.) indicates the specific interactions having place onlybetween novel Camellia products and substrate which mimics human skin.

Control experiments with pre-hydrated substrate demonstrated that afterirradiation the absorbance of Vitro-Skin® testing substrate (IMS TestingGroup, Milford, Conn.) was significantly decreased especially in therange 260-330 nm (FIG. 9). This effect reflects relatively lowphoto-stability of non-protected substrate, which was irradiated by highdose of broad-spectrum solar light.

Irradiation of the substrate with applied white tea extract initiatedchanges in absorbance spectra (FIG. 10) which is analogous with thespectral changes of non-protected Vitro-Skin® testing substrate (IMSTesting Group, Milford, Conn.). When contribution of substrate waseliminated, the spectra of non-irradiated and irradiated sampleindicated some similarities but did not demonstrate totally identicalbehavior. For example, the absorbance in the range 290-310 nm wasdecreased and wide peak at 360 nm started to form.

The irradiation of cell walls fraction extract (FIG. 11) led to similarchanges in the absorbance spectra especially with respect to the curveobtained after elimination (subtraction) of substrate contribution.

Although compositions of white tea extract and cell walls fractionextract are not identical, the pattern of irradiation-inducedmodifications in their spectra is quite similar. It should be noted thatboth white tea extract and cell walls fraction extract are not capableof fully protecting the substrate against destructive action ofirradiation and as result, the absorbance of Vitro-Skin® testingsubstrate (IMS Testing Group, Milford, Conn.) is decreased almost asmuch as at the condition when this substrate was not protected at all(FIG. 9). It is especially obvious in the range 250 nm-330 nm althoughat longer wavelengths some increase in the absorbance is observed.

The irradiation of membrane fraction extract produced very differenteffect on its absorbance spectra (FIG. 12). For example, irradiation didnot initiate any changes in spectral range 250-285 mm. As discussed,this particular range of spectra was very significantly impacted by thedestruction of irradiated Vitro-Skin® testing substrate (IMS TestingGroup, Milford, Conn.) and therefore comparison of the spectra suggestthat the destruction of the substrate was completely prevented by thepresence of membrane fraction extract on its surface.

However, certain changes in membrane fraction extract spectra wereregistered. For example, the absorbance was slightly decreased in therange 290-320 nm and it was accompanied with small increase ofabsorbance at longer wavelengths. It should be especially pointed out,that membrane fraction extract was proven to be very effective in therange of spectra where nucleic acids and aromatic amino acids havecharacteristic peaks at 260 nm and 280 nm subsequently. Thus, effect ofmembrane fraction extract allows the use of this product as aprospective UV protective ingredient for topical applications.

After irradiation the serum fraction was demonstrating certain changesin its absorbance spectra (FIG. 13).

Generally, these changes can be described as slight decrease of theabsorption in the range 250-340 nm and slight increase of the absorptionin the range 340-450 nm. It should be noted, that elimination of thepossible contribution provided by photo-destruction of Vitro-Skin®testing substrate (IMS Testing Group, Milford, Conn.) (see red curve onspectra above the initial spectra of non-irradiated serum fraction)clearly indicated that substrate was effectively preserved by theapplication of serum fraction on its surface.

Observations and Conclusions. The above results clearly show that thebest of conventional Camellia products—white tea extract—providesrelatively weak protection against the destructive action of UVirradiation. The cell walls fraction extract demonstrated propertiessimilar to white tea extract, but membrane fraction extract and celljuice serum have much more potent UV protective properties.

The UV protection properties of Camellia products were found to beincreasing in the following sequence: white tea extract=cell wallsfraction extract>cell juice serum>membrane fraction extract.

It should be noted, that spectral properties of Camellia products andpattern of the changes of these properties after UV irradiation providestrong evidence that compositions of constituents in white tea extract(control), cell walls fraction extract, membrane fraction extract andcell juice serum all differ and display unique activities. This is ofparticular interest in the case of novel Camellia compositions where theUV activity described above cannot be attributed to polyphenols as it iswith white tea extracts.

Example 10 Comparative Evaluation of Camellia Products: An Overview

Examples 10 through 19 describe methods, results, and analyses relatingto experiments conducted to evaluate the range of biological activitiesrelated to the modulation of cell functions by the bioactivecompositions from Camellia sinensis of the present invention. Theprimary objective was to evaluate the range of biological activities ofproducts obtained from the methods of the present invention and comparethese with activities of the best product obtained by conventional(traditional) tea technology—white tea extract, which was explored as apositive control and compared with the following bioactive compositionsof the present invention: (1) cell walls fraction extract of freshleaves (composition A, as referenced herein); (2) membrane fractionextract (composition B, as referenced herein); and (3) cell juice serum(composition D, as referenced herein).

The tests were conducted to evaluate the effect of these Camelliabioactive compositions on growth patterns of three human cell lines: amyeloid line with characteristics of monocytic leukemia cells (Mono Mac6) and two breast cancer lines with characteristics of early stages ofthe malignancy in vivo (MCF-7) and a more highly invasive, metastaticand estrogen insensitive line with characteristics of advanced cancer(MDA-MB-435S).

It was found that conventional white tea extract demonstrated a certaininhibitory effect on metabolic activity of some tumor cells. However,the extent of such inhibition was not significant for all types oftested cells and even when such inhibition was detected, it wasgenerally not complete but rather minimal or modest. The cell wallsfraction extract demonstrated properties similar to properties to thoseof white tea extract.

It is noteworthy that both of the cell juice derivatives: membranefraction extract and cell juice serum, were much more potent inhibitorsof metabolic functions of all tested cell lines which were cultured inthe presence and absence of different stimuli. For example, membranefraction extract clearly demonstrated greater inhibition potency and itseffect could be reliably measured at a dose of 0.001%. The cell juiceserum demonstrated a complex response: stimulation at a lower dose andinhibition at a high dose.

It should be noted that rather than inducing necrotic cytolysis,membrane fraction extract and cell juice serum appear to initiate apathway of programmed, or apoptotic cell death in the tumor cells. Theexperimental data indicate that this pathway is attributed to loss ofmitochondrial function and may require 24 to 48 hours of exposure to bedetected.

As a consequence of exposure to bioactive compositions, the metabolicfunction of all tested tumor cell lines: MCF-7, a model of early stagehuman breast cancer, MDA-MB-435S, a model of advanced breast cancer, andMono Mac 6, a model of monocytic leukemia, was inhibited, mosteffectively by the membrane fraction extract and, less potently and moreselectively, by the cell juice serum. Remarkably, the white tea extractand cell walls fraction extract were proven to be inactive or much lesspotent than the above compositions B and D. This trend was clearlyproven for the cells tested under different conditions: MCF-7 cells inthe absence and in the presence of transforming growth factor,MDA-MB-435S cells and both stimulated and non-stimulated monocytic MonoMac 6 cells.

These results provide strong evidence of the ability of the method ofthe present invention to drastically increase the potency of Camelliaplants and produce very impressive novel products demonstratingactivities, which were not identified for even the best product ofconventional tea technology.

Effects of the Camellia fractions of the present invention oncell-mediated proteolytic activities have implications for inflammatorytissue injury as well as tumor invasion and metastasis. Thus, breastcancer cells and leukemia cells clearly can be suggested as prospectivetargets for the bioactive compositions of the present invention, mostnotably, the membrane fraction extract. It should be noted that it waspreviously shown that the colon carcinoma-derived cell line COLO 205releases significant levels of MMP-2, which is then activated by atrypsin-like enzyme also secreted by the cells. This is also one ofpotential targets for the Camellia fractions of the present invention,based on results with Mono Mac 6 cells.

From these studies it has been concluded that the present invention'sbioactive compositions isolated from fresh Camellia have activitiesresult in impressive modulation of key cell functions. The effects thathave been observed could have valuable applications ranging frompersonal care products to nutraceuticals and potentiallypharmaceuticals.

It should also be noted that the present invention's very potentbioactive compositions are not single purified components, but ratherisolated complexes of constituents. Further fractionation of membranefraction extract (composition B) and cell juice serum (composition D)could yield extremely potent ingredients for the growing market ofnatural pharmaceuticals.

Example 11 Comparative Evaluation of Camellia Products: TestedCompositions

The following bioactive compositions were used in the experimentsdescribed in Examples 10 through 19:

-   -   (1) Positive Control: White tea extract which was prepared        according to the procedure described in Examples 1 and 4.    -   (2) Composition A: A cell walls fraction extract of fresh leaves        of Camellia which was prepared according to procedure described        in Examples 1 and 4.    -   (3) Composition B: A membrane fraction extract obtained from        freshly processed leaves of Camellia and prepared according to        procedure described in Examples 1 and 4.    -   (4) Composition D: Cell juice serum of freshly processed leaves        of Camellia and prepared according to the procedure described in        Examples 1 and 4.

The above products were obtained from the same lot of fresh Camellia toprepare the conventional white tea extract and three “parallel” productsof the present invention (compositions A, B and D).

There are a number of reports in the literature, which suggest thatextracts of Camellia leaves have a range of biological activities,primarily attributed to the significant concentrations of polyphenolictannins that form during the curing process. These polyphenols, as wellas lower molecular weight precursors to the polymeric tannins such asepigallocatechin-3-O-gallate (EGCG), have been reported to displaypotent antioxidant activities. There is a growing number of publicationssuggesting not only antioxidant, but also anti-angiogenic,anti-bacterial, anti-neoplastic, anti-inflammatory, anti-mutagenic,anti-septic, and detoxifying properties of teas prepared from driedleaves of Camellia. Not all of the above properties have been proven toconfer statistically significant benefits. Only some of them have beenconfirmed in comprehensive studies using multiple testing systems.

As past reference, it should be pointed out that, from past experiencewith bioactive compositions isolated from a number of fresh plantsources other than Camellia using the present invention's technology,such compositions were proven to be much more potent than conventionalproducts isolated from the same dried plants using a number ofparameters as was previously described in the U.S. Patent ApplicationPublication No. 2003/0175235, which is hereby incorporated by referencein its entirety). For example, in other types of plants (Medicagosativa, Hordeum vulgare, Lavandula angustifolia, Calendula officinalisand Salvia officinalis), several impressive biological activities ofcompositions prepared using the method of the present invention havebeen identified and evaluated, including high anti-elastase andanti-gelatinase B (MMP-9) activities, novel modulation of the neutrophilrespiratory burst, and significant superoxide scavenging activitytowards reactive oxygen species. Other than scavenging activity, theseactivities are not likely to be ascribed to mixtures of polyphenolsalone.

Thus, it was especially interesting to explore a more comprehensiveapproach to compare the range of activities, which could be detected inthe Camellia compositions of the present invention with the activitiespresent in an extract obtained from the same dried plant usingconventional (traditional) Camellia technology. Accordingly, modulationof functions in living mammalian cells by the cell walls fractionextract (composition A), membrane fraction extract (composition B) andcell juice serum (composition D) prepared from freshly collected leavesof Camellia have been assayed. These compositions have been compared toextract of conventional white tea prepared from dried Camellia leaves.

It should be noted that, according to multiple studies of conventionalCamellia products, the white tea extract demonstrated higher specificactivities and therefore a preparation of this sort was selected as arepresentative positive reference control for comparison with the novelCamellia products of the present invention.

Example 12 Comparative Evaluation of Camellia Products: Rationale forSelection of Cell Lines

As a test system for modulation of cell functions, two human breastcarcinoma-derived lines were used as models of neoplastic cells (MCF-7and MDA-MB-435S), and a human monocytoid line (Mono Mac 6) was used as amodel of inflammatory cells. The above cell lines are described inExample 21.

MCF-7 is considered a model of early or less de-differentiated breastcancer. The line still retains estrogen sensitivity and has a relativelylow invasive phenotype; its capacity to metastasize in immunodeficientanimal models is quite modest. In previous studies, the MCF-7 cell linehas been shown to display a characteristic response to TransformingGrowth Factor-β(TGF-β): after culture for 24 hours in the presence ofTGF-β, the cells secrete increased levels of the Matrix MetalloProteinase (MMP) family of proteolytic enzymes and the pro-angiogenicfactor Vascular Endothelial Growth Factor (VEGF), two different markersof enhanced invasiveness and metastatic potential. This response toTGF-β is a mark of tumors and some tumor cell lines, in contrast togrowth arrest, which is induced in normal cells by the growth factor. Toevaluate the bioactive compositions of the present invention, MCF-7cells cultured in the absence and presence of TGF-β were used astargets.

The MDA-MB-435S line is more highly invasive, metastatic and estrogeninsensitive. This human carcinoma-derived cell line also shows somesensitivity to TGF-β, but even in the absence of the growth factor, itspontaneously releases higher levels of MMPs and VEGF than MCF-7,consistent with its use as a model of more advanced cancer. In presentevaluation the effects of bioactive compositions on MDA-MB-435S cellscultured only in the absence of TGF-β were examined.

The human monocytoid line, Mono Mac 6, expresses a number of biomarkersconsistent with those of resting monocytes or macrophages, and respondslike human monocytes and macrophages to pro-inflammatory activatingstimuli such as Phorbol Myristate Acetate (PMA). The effects ofbioactive compositions on Mono Mac 6 cells cultured in the absence andpresence of PMA were examined, to serve as models of resting andactivated monocytes/macrophages.

Thus, the selected combination of the cell lines described aboveprovides a reliable foundation for evaluations of anti-tumor andanti-inflammatory potencies of Camellia bioactive compositions. Paralleltesting of selected cell lines with a number of functional probesprovides the opportunity to draw more valuable conclusions concerningthe activities of products and their mechanism of action-thaninvestigation of the responses of a single test target or targets havingsimilar sensitivities or similar responses to certain stimuli.

Example 13 Comparative Evaluation of Camellia Bioactive Compositions:Rationale for Selection of Assays

Initial evaluations were based on two viability assays and a probe ofcell functions (see Example 20, “Method 8”).

The first assay measures levels of the cytosolic enzyme, lacticdehydrogenase, which is liberated into the extracellular culture mediumonly when the cells lyse. Such loss of cell membrane integrity istraditionally considered to be a sign of necrotic cell death andreflects the cytotoxicity pattern.

The second assay measures mitochondrial dehydrogenase activity asreflected by the reduction of a tetrazolium salt to its coloredformazan. When the MTS reagent (a tetrazolium salt) is applied to livingcells, it is converted to an intensely colored compound (formazan). Lossof mitochondrial dehydrogenase activity can also be associated with celldeath, but is typically a marker for the early steps in a programmedcell death, or apoptotic, pathway in which cell membrane integrity isgenerally retained well after the nucleus has condensed and themitochondria have ceased to function.

The leakage of lactic dehydrogenase indicates complete loss of viabilityassociated with cytolysis, while decreased reduction of tetrazoliumsalts indicates loss of mitochondrial activity, but not necessarilyirrevocable loss of cell membrane integrity or viability.

As an additional probe of cell functions, the effects of the Camelliabioactive compositions on levels of proteinases secreted by the Mono Mac6 line have been examined (see Example 20, “Method 9”). In previousstudies with this cell line, it was observed that, after incubation withPMA, Mono Mac 6 cells secrete two so-called gelatinolytic matrixmetalloproteinases, MMP-2 (gelatinase A) and MMP-9 (gelatinase B). TheseMMPs are also secreted by a number of tumors and by their surroundingstroma, and are implicated in inflammatory tissue injury as well astumor invasion and metastasis. It was also previously shown that someagents under development as anti-inflammatory and anti-tumor drugs (theagents that have been investigated are known to diminish inflammatorytissue destruction as well as invasion and metastasis of tumor celllines) appear to reduce the levels of the MMPs produced by cells inaddition to any direct inhibition of MMP proteolytic activity. Theobjective in these studies was to evaluate the possibility that theCamellia bioactive compositions of the present invention might have asimilar capacity to diminish levels of MMPs released by activated MonoMac 6 cells.

Thus, the selected assays will allow one to reliably evaluate a broadspectrum of metabolic processes and effectively obtain important data,which might reveal mechanisms of action triggered by certain Camelliabioactive compositions.

Example 14 Comparative Evaluation of Camellia Products: Effects ofCamellia Bioactive Compositions on Breast Tumor Cell Lines

Throughout these studies, mitochondrial function was measured solelythrough assays of reduction of the tetrazolium salt MTS to its formazan.It should be noted that some intrinsic capacity of higher concentrationsof the Camellia compositions of the present invention have been observedto reduce MTS directly in the absence of any viable cells, and in allthe results reported here, such background formation of formazan in theabsence of cells has been subtracted from the levels of reductaseactivity observed in the presence of the cells.

FIGS. 14 through 21 illustrate the magnitude of the reductase activityof MCF-7 cells, cultured in the absence and presence of 5 ng/ml TGF-β,and MDA-MB-435S cells, cultured only in the absence of TGF-β, at 24hours and 48 hours after the addition of various doses of each of thefour Camellia compositions, ranging from 0.01% or 0.02% (w/v, finalconcentration in the culture medium, based on dry weight of the solidsin the Camellia compositions) to 0.0001%.

Example 15 Comparative Evaluation of Camellia Bioactive Compositions:MCF-7 Cells

In the absence of TGF-β, the highest tested concentration (0.01%) of thecomposition A and white tea extract (positive control) had a markedeffect on MTS reduction by MCF-7 cells. At that concentration there wassignificant but incomplete inhibition of reductase activity (˜50-70%inhibition) after 24 hours of exposure to the Camellia composition A.The similar inhibition of reductase activity was detected after 24 hoursof exposure to white tea extract.

In contrast, the two Camellia bioactive compositions (membrane fractionextract and cell juice serum) prepared from Camellia cell juice weremore potent inhibitors of reductase activity in MCF-7 cells in theabsence of TGF-β. The membrane fraction extract (composition B)resembled white tea extract in dose dependence, except for somewhatgreater potency, producing virtually complete inhibition at 0.01%, thehighest dose tested. The cell juice serum (composition D) also producedvirtually complete inhibition at 0.01%, but at the lower dose of0.0025%, there was some evidence of stimulation of reductase activity.Lower doses of composition D were without significant effect.

When MCF-7 cells were cultured in the presence of the growth factorTGF-β, their sensitivity to the Camellia compositions was significantlyaltered. After 24 hours or 48 hours of exposure to the cell wallsfraction extract and white tea extract, there was no evidence of eithera stimulatory or an inhibitory effect on reductase activity at any dose,except for a modest inhibition of less than 20% at the highest dose(0.01%) of white tea extract.

In contrast, exposure of TGF-β-treated MCF-7 cells for 24 hours to themembrane fraction extract at a dose of 0.02% resulted in 70% inhibitionof reductase activity, and after 48 hours, reductase activity wasvirtually completely abated. More modest inhibition could be detected atlower doses of membrane fraction extract after 24 hours, but after 48hours, a marked activation of reductase activity was detected. The sameactivation of reductase activity by low doses of the cell juice serum aswell as marked inhibition at the highest dose (0.02%) was detected after48 hours of exposure, but this composition had only minimal effect onreductase activity in TGF-β-treated MCF-7 cells after the more limitedexposure of 24 hours, regardless of dose.

In evaluation, there was no detection of significant release of lacticdehydrogenase into the culture medium of MCF-7 cells exposed for 24hours to even the highest doses of any of the Camellia compositions. Itwould appear that, the loss of mitochondrial function in these cells isnot accompanied by a necrotic lysis of the cells. If the cells are infact dying during the first 48 hours of exposure, it is more likely thata programmed cell death, or apoptotic, pathway has been initiated. Thisconclusion is supported by light microscopic observations, which revealthat there is some rounding of the cells, but no formation of debris ormembrane fragments during the course of the exposures.

Thus, inhibition of mitochondrial function appears to be a predominantmode of action of all tested Camellia products, which did notdemonstrate cytotoxity or necrosis as indicated by levels of releasedlactic dehydrogehase.

Example 16 Comparative Evaluation of Camellia Bioactive Compositions:MDA-MB-435S Cells

The pattern of response of MDA-MB-435S cells to the Camelliacompositions was similar to that of MCF-7 cells in that the most potentcompositions were composition B and D, with the membrane fractionextract (composition B) showing somewhat greater potency than the celljuice serum (composition D). Only the membrane fraction extract producedmarked inhibition of reductase activity after only 24 hours of exposureInhibition reached −70% of control reductase values at the highest doseof 0.01%, but a modest inhibition of −10% could be reliably detected ateven the lowest dose of remarkable concentration −0.0001%. The othertested compositions had only modest inhibitory effects at 24 hours ofexposure, and only at the higher doses tested.

After 48 hours of exposure, reductase activity was inhibited in adose-dependent fashion in the presence of each of the compositions, butthe potency at the highest dose of the compositions did not reach near100% inhibition, except for the membrane fraction extract. Thiscomposition inhibited reductase activity by −50% at 0.001% after 48hours. The white tea extract and cell walls fraction extract also hadsignificant inhibitory activity against MDA-MB-435S cells after 48 hoursof exposure, which was actually greater than that of the cell juiceserum. No doses of any of the preparations induced activation ofreductase activity in this cell line, regardless of duration ofexposure.

It should be noted that any impact on highly invasive, metastatic andestrogen insensitive line MDA-MB-435S is rare to observe after only 24hours. Thus, the effect of composition B after 24 and 48 hours is ratherremarkable and indicates that this preparation has significant activity.

Example 17 Comparative Evaluation of Camellia Bioactive Compositions:Effects of Camellia Compositions on Monocytoid Cells

Mitochondrial Dehydrogenase Activity: Certain of the trends revealed bythe preliminary studies on the breast tumor cell lines have proved to beconsistent with the response of Mono Mac 6 cells to the four testedCamellia compositions. The membrane fraction extract (composition B) andcell juice serum (composition D) were more potent inhibitors of MTSreductase activity in this inflammatory cell line than the cell wallsfraction extract (composition A) and white tea extract (positivecontrol), and the membrane fraction extract (composition B) clearly hadthe greatest inhibitory potency. The effects on MTS reductase in cellswhich were left unstimulated and those which were stimulated with 10 nMPMA were examined, and reductase activity after 24 and 48 hours ofexposure to the Camellia compositions was evaluated. The effects ofthese compositions on MTS reductase activity in Mono Mac 6 cells areshown in FIGS. 22 through 33.

The white tea extract showed a weak but dose-dependent inhibition ofreductase activity after 48 hours of exposure to PMA-stimulated cells;there was no significant loss of reductase activity regardless of thedose or length of exposure in the absence of PMA, nor was there anyeffect of any dose after 24 hours of exposure to PMA-treated cells. Thecell walls fraction extract had no effect on Mono Mac 6 cells regardlessof dose or time of exposure and regardless of whether the cells wereunstimulated or stimulated with PMA.

The cell juice serum of fresh Camellia leaves inhibited unstimulatedMono Mac 6 cell reductase activity modestly in a dose dependent fashionafter 24 or 48 hours of exposure. The maximum inhibition at the highestdose of 0.01% (w/v, final concentration in the culture medium, based ondry weight of solids in the starting preparation) was only ˜20-30% ofthe control activity. Inhibition of PMA-stimulated cells reached 50% ofcontrol activity but only at the highest dose of composition D (0.01%),and only after 48 hours of exposure.

The membrane fraction extract of freshly harvested Camellia (compositionB) proved to be the most potent of the tested preparations in inhibitingMono Mac 6 cell reductase activity as it had toward the breast tumorcell lines. Effects of PMA stimulation or duration of exposure to thecomposition had little effect on inhibition, which was dose-dependent inthe presence or absence of PMA and was roughly the same after 24 hour or48 hour exposure. Reductase activity was inhibited by −70% in thepresence of PMA and by −80-90% in the absence of PMA at the highest doseof 0.02%, but lower levels of inhibition (−15%) could be reliablymeasured at a dose of 0.001%. Measurements of release of cytosolicenzymes have not been undertaken to confirm that the loss of reductaseactivity is not associated with necrotic cytolysis, but no evidence ofmembrane fragmentation could be seen by light microscopic examination ofMono Mac 6 cells exposed to any of the bioactive compositions at 0.02%for 48 hours. Moreover, as shown below, the cells appear still capableof secreting at least one MMP under conditions in which reduction of MTSis markedly diminished.

These results suggest that in these cells, as well as the breast tumorcell lines, the loss of reductase activity is associated with arelatively selective loss of mitochondrial function and can reflectinitiation of a pathway of programmed cell death or apoptosis.

Secretion of MMPs. Two different assays have been used to measure thelevels of two gelatinases, MMP-2 (gelatinase A) and MMP-9 (gelatinaseB), released by Mono Mac 6 cells. These MMPs have been implicated ininflammatory tissue damage as well as tumor invasion and metastasis.Employed enzyme-linked immunosorbent assays (ELISAs) for MMP-2 and MMP-9were first used to estimate total levels of the two enzymes in theculture medium of Mono Mac 6 cells cultured for 48 hours in the presenceof 10 nM PMA and different doses of the three Camellia bioactivecompositions and positive control.

This cell line secretes only MMP-2 when it is unstimulated, but secretesboth MMP-2 and MMP-9 when it is activated. (Levels of MMPs releasedafter 24 hours are usually too low to be reliably detected).

The results of the ELISA measurements are shown in FIGS. 34 through 37.As was observed for the effects of cell walls fraction extract and whitetea extract on MTS reduction by Mono Mac 6 cells, there was nosignificant change in levels of secreted MMP-2 or MMP-9 at any dose ofthese extracts. At the highest dose (0.01% w/v) of the membrane fractionextract and cell juice serum, the levels of MMP-2 were observed to bediminished, with the membrane fraction extract exhibiting the greatestpotency at this dose. It should be noted, that an apparent slightstimulation of MMP-2 release was observed at the next highest doses ofcomposition B (0.001%) and composition D (0.002%). This stimulation isreminiscent of the stimulation of MTS reductase activity in TGF-βtreated MCF-7 cells at similar doses of these compositions.

The dose-dependent diminution of MMP-2 levels detected by ELISA was notparalleled by the effects of membrane fraction extract and cell juiceserum on MMP-9 levels. These levels were increased (apparently markedlyso by cell juice serum) at the highest doses, but were unchanged at thelower doses tested. The detection of unchanged or increased levels ofMMP-9 secreted by Mono Mac 6 cells exposed for 48 hours to doses ofCamellia preparations which produced significant inhibition of MTSreductase activity after only 24 hours, is further evidence that theloss of mitochondrial function in Mono Mac 6 cells exposed to themembrane fraction extract or cell juice serum of Camellia does notreflect necrotic cytolysis, in which case MMP secretion would haveabruptly ceased.

As further evidence of the effects of the Camellia compositions on MMPsecretion by Mono Mac 6 cells, the technique of gelatin zymography wasused to examine the culture media collected as described above for theELISA measurements. In this method, the culture media are firstsubjected to electrophoresis in gelatin-impregnated polyacrylamide gelsin the presence of Sodium Dodecyl Sulfate (SDS-PAGE) to separate theproteins on the basis of molecular weight. The SDS is then washed out ofthe gels to allow at least a portion of any enzymes present to renatureand the gels are incubated in a medium, which maximizes MMP activity.MMPs dissolve the gelatin wherever they may be present. Aftervisualizing the undigested gelatin in the bulk of the gels with aprotein stain, the gels are scanned, with the MMPs appearing as clearzones against the stained background. Negative images have beenpresented here, so that the MMPs appear as dark zones against a lightbackground.

It should be noted that MMPs are secreted by most cells as inactiveprecursors, which are then activated extracellularly. However, becauseof the denaturing and renaturing sequence employed in zymography, eventhe so-called inactive pro-forms of the MMPs acquire gelatinolyticactivity and produce clear zones. FIGS. 34 through 37 illustrate thenegative images of gelatin zymograms of culture media collected after 48hour exposure of Mono Mac 6 cells to the different Camellia bioactivecompositions, along with culture medium collected from cells cultured inthe absence (U) or presence (S) of 10 nM PMA but in the absence ofCamellia compositions.

The effects of composition A and positive control were evaluated onlyfor the lowest dose (0.0001%, “10”) and the highest dose (0.01%, “hi”)of the preparations, whereas the effects of compositions B and D werealso evaluated at the intermediate dose of 0.001% (“med”). It isapparent from the four panels that Mono Mac 6 cells release only MMP-2(˜67 kD) in the absence of PMA, and this enzyme is found predominantlyin the pro-form. Treatment with 10 nM PMA results in induction of MMP-9secretion (˜0.92 kD), as well as further proteolytic activities whichconvert significant levels of the pro-forms of the two MMPs to theirslightly lower molecular weight active forms.

Consistent with the ELISA results, exposure of PMA-stimulated Mono Mac 6cells to cell walls fraction extract and white tea extract had nodetectable effect on the levels of either the pro- or active forms ofeither of the two MMPs visualized by gelatin zymography. In contrast,exposure to the highest dose of compositions B and D resulted in markeddiminution of the levels of MMP-2 visualized by gelatin zymography, butno apparent change in the levels of MMP-9.

The appearance of both pro- and active forms of MMP-9, as well as thefaint, but recognizable, band corresponding to the active form of MMP-2seen in the media collected from cells treated with the highest dose ofcompositions B and D, suggests that the effects of these compositionsare primarily on modulation of release of MMP-2 and do not involveadditional effects on the MMP activation mechanisms in these culturedcells.

Example 18 Comparative Evaluation of Camellia Bioactive Compositions:Summary of Results

The experimental data indicate that Camellia bioactive compositionstrigger a dose-dependent loss of MTS reductase activity, which isgenerally attributed to loss of mitochondrial function. This inhibitionmay require as long as 48 hours of exposure to be detected and at leastfor the first 24 hours, there is no measurable release of cytosolicenzymes, suggesting that rather than inducing necrotic cytolysis, thebioactive compositions initiate a pathway of programmed, or apoptotic,cell death in the tumor cells.

The differences in the time- and dose-dependence of the response ofMCF-7 cells and MDA-MB-435S cells, and the effects of TGF-β treatment ofthe MCF-7 cells, all point to a somewhat increased resistance of themore invasive and metastatic phenotypes to white tea extract, cell wallsfraction extract, and to some degree, cell juice serum, as evidenced bythe relatively modest loss of reductase activity within the first 24hours of exposure. However, the trend of greater potency of the membranefraction extract is evidenced by its capacity to inhibit MTS reductaseactivity in TGF-β-treated MCF-7 cells, as well as MDA-MB-435S cellswithin 24 hours.

The effects of tested Camellia bioactive compositions on the Mono Mac 6cell line, a model of human monocytes/macrophages, have certainsimilarities to the effects observed on breast tumor cell lines. Basedon the absence of lactic dehydrogenase in the culture medium of thebreast tumor cell lines and the presence of normal to increased levelsof secreted MMP-9 in the culture medium of Mono Mac 6 cells, it has beenconcluded that these compositions do not induce necrotic cytolysis, evenat the highest dose tested (0.01% w/v).

However, the two bioactive compositions (membrane fraction extract andcell juice serum) induce a dose-dependent inhibition of mitochondrialreductase activity, which reflect initiation of an apoptotic pathway ofprogrammed cell death in Mono Mac 6 cells. Furthermore, exposure ofthese inflammatory cells to the membrane fraction extract and cell juiceserum results in selective diminution in the levels of the gelatinolyticenzyme, MMP-2 (gelatinase A). The gelatin zymography indicates thatmechanisms of “pro-form” or zymogen activation are unaffected by theCamellia bioactive compositions, so it is highly unlikely that thediminished levels MMP-2 in the medium reflect enhanced proteolyticdestruction.

Thus, the metabolic activity of all tested cell lines (i.e., a model ofearly stage human breast cancer, a model of advanced breast cancercells, and a model of monocytic leukemia) was effectively inhibited bythe membrane fraction extract (composition B) and, in most of cases, thecell juice serum (composition D). Remarkably, the extract of cell wallstea (Composition A) and white tea extract (positive control) were provento be inactive or much less potent than the above compositions B and D.

This trend was clearly proven for all tested MCF-7 human cancer cells inthe absence and in the presence of transforming growth factor, forMDA-MB-435S advanced human breast cancer cells, and for stimulated andnon-stimulated monocytoid Mono Mac 6 cells. The data related to thesummary of testing and evaluation of bioactive Camellia compositions arepresented in Table 15.

TABLE 15 Summary of Testing and Evaluation of Bioactive CamelliaCompositions Extract of White Tea Extract of Cell Membrane Time ofExtract Walls Fraction Fraction Cell Juice Serum Cell Line and ModelStimuli Cultivation (Positive Control) (Composition A) (Composition B)(Composition D) Human Cancer Cells 24 hours Modest Modest Strong ModestMDA-MB-435S Inhibition Inhibition Inhibition Inhibition Advanced Breast48 hours Significant but Significant but Complete Significant but CancerIncomplete Incomplete Inhibition Incomplete Inhibition InhibitionInhibition Human Cancer Cells 24 hours Significant but Significant butComplete Complete MCF-7 Incomplete Incomplete Inhibition InhibitionInhibition Inhibition Early Breast Cancer TGF-β 24 hours Modest NoEffect Significant but Complete Inhibition Incomplete InhibitionInhibition 48 hours Modest No Effect Stimulation at Stimulation atInhibition Lower Dose and Lower Dose and Complete Significant butInhibition at Incomplete High Dose Inhibition at High Dose HumanLeukemia 24 hours Modest Modest Complete Pronounced Cells Mono Mac 6Inhibition Inhibition Inhibition Inhibition 48 hours No Effect No EffectSignificant but Pronounced Incomplete Inhibition Inhibition InflammationPMA 24 hours No Effect Modest Significant but Significant but InhibitionIncomplete Incomplete Inhibition Inhibition 48 hours Modest No EffectSignificant but Significant but Inhibition Incomplete IncompleteInhibition Inhibition

Table 15 shows that abilities of Camellia preparation to modulate cellfunctions in a dose-dependent manner is increasing in the followingorder: white tea extract=cell walls fraction extract>cell juiceserum>membrane fraction extract. The experimental data suggests that,novel bioactive Camellia compositions prepared by processing of freshplant tissue into cell juice derived membrane fraction extract(composition B) and cell juice serum (composition D) do not trigger anyoutright necrotic toxicity towards the cells.

Therefore, the technology of the present invention displays the abilityto drastically increase the potency of Camellia bioactive compositionsand to produce very impressive novel products demonstrating activitieson viable human cells which were not demonstrable in the best products(for example, white tea extract) produced by conventional Camelliatechnology.

Example 19 Comparative Evaluation of Camellia Bioactive CompositionsImplications for Future Studies

Effects of the Camellia bioactive compositions of the present inventionon cell-mediated proteolytic activities have implications forinflammatory tissue injury as well as tumor invasion and metastasis.Thus, breast cancer cells and monocytic leukemia cells clearly can besuggested as prospective targets for the Camellia bioactive compositionsof the present invention, most notably, composition B (membrane fractionextract). It was previously shown that the colon carcinoma-derived cellline COLO 205 releases significant levels of MMP-2, which is thenactivated by a trypsin-like enzyme also secreted by the cells. This typeof tumor cell is one of a number of potential targets for the Camelliabioactive compositions of the present invention, based on results withMono Mac 6 cells.

From these studies, one can be confident that the bioactive compositionsisolated from fresh Camellia of the present invention have significantactivities, which result in impressive modulation of key cell functions.The effects that have been observed have valuable applications rangingfrom personal care products to nutraceuticals and potentiallypharmaceuticals.

Example 20 Protocols Used for Determining Certain Characteristics ofBioactive Compositions

The following are various methods used for determining certaincharacteristics of Bioactive Compositions. These methods are referencedthroughout the above Examples. References below to the “tested products”or the “test samples” refer to Bioactive Compositions.

Method 1: Method for Determination of Solid Content. The procedure fordetermination of solid content included evaporation of the testedbioactive composition in the water bath at 100° C. until completeevaporation of water, oven storage of the sample at 105° C. for 3 hours,cooling to room temperature, and immediate determination of the weightof the container with solid matter.

Method 2: Method for Determination of Non-Volatile Residue. Theprocedure for determination of non-volatile residue included ovenstorage of the tested bioactive composition at 105° C. for 5 hours,cooling, and immediate determination of the weight of the container withsolid matter.

Method 3: Method for Determination of L*a*b* Values. The procedure fordetermination of L*a*b* values utilized Hunter Labscan fixed geometrycolorimeter with measuring geometry of 0°/45°. Standard illuminant D₆₅with viewing window facing upward was used. The container with testedbioactive composition was placed on viewing window and measured throughthe bottom. The following CIELAB equations were used:C*=(a* ² +b* ²)^(1/2)DE*=[(DL)²+(Da*)²+(Db*)²]^(1/2)DH=[(DE*)²−(DL*)²−(DC*)²]^(1/2).

Method 4: Method for Determination of Total Carotenoids Content andLutein Content. The tested bioactive compositions were extracted withacetone. After homogenization and vacuum filtration, all extracts weresaponified with 30% potassium hydroxide in methanol. The carotenoidswere successively extracted from bioactive compositions with petroleumether. After additional treatment and re-solubilization in ethanol, allsamples were measured at 446 nm.

In order to determine the lutein content, an additional dried samplefrom each sample extraction was used for high performance liquidchromatography (“HPLC”) analysis. The dried sample was re-solubilized inMTBE and methanol. The reverse phase HPLC system with (250×4.60 mm I.D.)5 μm C₁₈ column (“Vydac”) was used. The identity of lutein was conformedby the co-chromatography of an authentic standard. The molarabsorptivity coefficient for lutein in ethanol is 144,800 cm⁻¹ mol⁻¹.

Method 5: Method for Determination of Elastase Inhibitory Activity. Theelastase inhibitory activity of tested bioactive compositions wasdetermined using the assay, which employs neutrophil elastase (apurified enzyme preparation produced by “Elastin Products”) andsynthetic peptide soluble substrateMethoxysuccinyl-Ala-Ala-Pro-Val-p-Nitroanilide produced by “Sigma”.Enzymatic cleavage of the substrate results in generation of increasingyellow color over time (405 nm); the rate of color generation isdiminished by increasing concentrations of tested bioactive compositionscontaining inhibitory activity. Analysis of the concentration dependenceof inhibition permits quantitation of the potency of the inhibitoryactivity, expressed as that concentration of dry matter within eachtested bioactive required to achieve 50% inhibition (IC₅₀), but alsoprovides information relating to the mode of inhibition.

For the determination of IC₅₀, the concentration of elastase was 2.5μg/ml and concentration of substrate was 150 μM. For the determinationof K_(i), the concentrations of substrate were 100 μM and 200 μM.

Method 6: Method for Determination of Gelatinase B (MMP-9) InhibitoryActivity. The commercially distributed assay (MMP-9 Activity ELISAproduced by “Amersham Pharmacia”), which captures Gelatinase Bspecifically onto multiwell microplates by immune recognition, was usedafter other proteinases were washed away. The enzymatic activity wasdetected at 405 nm by hydrolysis of a low molecular weight syntheticsubstrate for Gelatinase B: APMA. Analysis of the concentrationdependence of inhibition was used to determine the potency of testedbioactive composition dry matter.

Method 7: Method for Determination of Superoxide Scavenging Activity.The enzymatic system, which uses xanthine oxidase (a purified enzymepreparation produced by “Sigma”), was used to generate superoxide anionsin high yield and in a controlled fashion. The conversion of xanthine tohydroxanthine by this enzyme generates amounts of superoxide anions andreduction of ferricytochrome c to ferrocytochrome c was used as asensitive measure of superoxide levels. The measurements offerrocytochrome c level (550 nm), when tested bioactive compositionswere added to the reaction system, allow for determination of theirsuperoxide scavenging activity. The final concentrations per well werefor cytochrome c 75 μM, xanthine 425 μm/L, and xanthine oxidase 10mU/ml.

Method 8: Method for Determination of In Vitro Toxicity and Apoptosis.CellTiter 96 AQ_(ueous) One Solution Cell Proliferation Assay andCytoTox 96 Non-radioactive Cytotoxicity Assay and subsequent protocolswere explored (both assays produced by Promega Corporation, Madison,Wis.).

The first assay is a colorimetric method for determining the number ofviable cells which explores a tetrazolium compound(3-(4,5-dimethylthianzol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt; MTS and a electron coupling reagent (phenazine methosulfate;PMS). MTS is bioreduced by cells into a soluble in tissue culture mediumformazan product that has an absorbance maximum at 490 nm. Theconversion of MTS into aqueous, soluble formazan is accomplished bydehydrogenase enzymes found in metabolically active cells and thequantity of formazan product is directly proportional to the number ofliving cells in culture.

The second assay quantitatively measures lactate dehydrogenase (LDH), astable cytosolic enzyme that is released upon cell lysis. Released LDHin cell culture supernatant is measured with a 30-minute coupledenzymatic assay, which results in the conversation of a tetrazolium salt(INT) into a red formazan product. The amount of color formed isproportional to the number of lysed cells.

Method 9: Method for Determination of Level of Enzymes Secreted byStimulated Cells. After incubartion with PMA, Mono Mac 6 cells secretetwo gelatinolytic matrix metalloproteinases, MMP-2 (gelatinase A) andMMP-9 (gelatinase B). The levels of these enzymes in the presence oftested bioactive compositions were determined by two-dimensional sodiumdodecyl sulphate polyacrylamide gel electrophoresis.

Example 21 Cell Lines Used for Testing Certain Bioactive Characteristicsof the Camellia Products

The cell line MDA-MB-435S which is considered a model of advanced breastcancer was obtained from American Type Culture Collection (ATCC NumberHTB-129). This cell line was cultivated at 37° C. in the following ATCCmedium: Leibovitz's L-15 medium with 2 mM L-glutamine supplemented with0.01 mg/ml insulin, 90%; fetal bovine serum, 10%.

The cell line MCF-7 which is considered a model of early or lessde-differentiated breast cancer was obtained from ATCC (Number HTB-22)was cultivated at 37° C. in the following ATCC medium: Minimum essentialmedium (Eagle) with 2 mM L-glutamine and Earle's BSS adjusted to contain1.5 g/L sodium bicarbonate, 0.1 mM non-essential amino acids and 1 mMsodium pyruvate and supplemented with 0.01 mg/ml bovine insulin, 90%;fetal bovine serum, 10%.

The cell line MonoMac6 (MM6, obtained from the German Collection ofMicroorganisms and Cell Cultures) which closely resembles adifferentiated human monocyte (Ziegler-Heitbrock et al., “Establishementof a Human Cell Line (Mono Mac 6) with Characteristics of MatureMonocytes,” International Journal of Cancer 41:456-461 (1988), which ishereby incorporated by reference in its entirety). Cells were maintainedin RPMI 1640, supplemented with 2 mM L-glutamine, 100 U/ml penicillin,100 μg/ml streptomycin, 1 mM sodium pyruvate, 10% FCS, nonessentialamino acids, 9 μg/ml insulin, and 1 mM oxalacetic acid. For assayconditions, 0.2% glucose was also added.

Example 22 Catechin Analyses of the Camellia Products

The cell walls fraction extract, the membrane fraction extract, and thecell juice serum of the present invention were analyzed for content ofvarious catechins. A white tea sample was used as a control. Thefollowing catechins were assayed: (−)-epigallocatechin; (+)-catechin;(−)-epicatechin; (−)-epigallocatechin gallate; (−)-gallocatechingallate; and (−)-epicatechin gallate.

The samples were extracted using 0.1% H₃PO₄ and sonication for about 15minutes. After centrifugation, the extract was injected on HPLC. C-18reverse phase column was used as the stationary phase. 0.1% phosphoricacid and acetonitrile were used as the mobile phases. The detection wasat 280 nm. The calculation is based on comparing areas of each catechinlisted with its pure standard. The results are shown in FIG. 38 andTable 16 (below).

TABLE 16 Catechin Content of Bioactive Camellia Compositions Averagemg/g based Average mg/g on 100% dry based on Sample Chemical Analyzedmatter product as is White Tea (−)-epigallocatechin 0.3 0.0033 Extract(+)-catechin 1.33 0.0146 (−)-epicatechin 0.15 0.0017(−)-epigallocatechin gallate 0.65 0.0071 (−)-gallocatechin gallate 0.0030.0000 (−)-epicatechin gallate 0.212 0.0023 Cell Walls(−)-epigallocatechin 0.00 0.0000 Fraction (+)-catechin 2.39 0.0201Extract (−)-epicatechin 0.01 0.0001 (−)-epigallocatechin gallate 0.010.0001 (−)-gallocatechin gallate 0.00 0.0000 (−)-epicatechin gallate0.006 0.0001 Membrane (−)-epigallocatechin 2.27 0.1552 Fraction(+)-catechin 8.96 0.6121 Extract (−)-epicatechin 0.60 0.0409(−)-epigallocatechin gallate 9.28 0.6340 (−)-gallocatechin gallate 0.010.0006 (−)-epicatechin gallate 2.33 0.1589 Cell Juice(−)-epigallocatechin 3.01 0.1714 Serum (+)-catechin 6.09 0.3465(−)-epicatechin 0.95 0.0539 (−)-epigallocatechin gallate 1.97 0.1120(−)-gallocatechin gallate 0.04 0.0024 (−)-epicatechin gallate 0.570.0325

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow.

1. A bioactive composition comprising an isolated bioactive fraction,wherein said isolated bioactive fraction is produced by a processcomprising: (i) providing fresh biomass of a Camellia sinensis plant,wherein said fresh biomass comprises fresh Camellia sinensis plantbiomass that has not undergone conventional tea processing; (ii)separating the fresh biomass of the Camellia sinensis plant into celljuice and a cell walls component, wherein said separating comprisesgrinding, macerating, and pressing the fresh biomass to separate thefresh biomass into the cell juice and the cell walls component; (iii)treating the cell juice under conditions effective to yield a bioactivefraction, said conditions comprising subjecting the cell juice tomicrowave treatment to induce coagulation, thereby yielding a coagulatedmembrane fraction and a non-coagulated portion of the cell juice,wherein said bioactive fraction is the coagulated membrane fraction; and(iv) isolating said bioactive fraction from the treated cell juice. 2.The bioactive composition according to claim 1 further comprising astabilizing agent.
 3. The bioactive composition according to claim 2,wherein said stabilizing agent is selected from the group consisting ofan emulsifier, a preservative, an antioxidant, a polymer matrix, andmixtures thereof.
 4. A bioactive topical formulation suitable fortopical application to a mammal, said bioactive topical formulationcomprising: a topically effective amount of the bioactive compositionaccording to claim 1 and a topically acceptable carrier.
 5. Thebioactive topical formulation according to claim 4, wherein thetopically acceptable carrier is selected from the group consisting of ahydrophilic cream base, a hydrophilic lotion base, a hydrophilicsurfactant base, a hydrophilic gel base, a hydrophilic solution base, ahydrophobic cream base, a hydrophobic lotion base, a hydrophobicsurfactant base, a hydrophobic gel base, and a hydrophobic solutionbase.
 6. The bioactive topical formulation according to claim 4, whereinthe bioactive composition is present in an amount ranging from betweenabout 0.001 percent and about 90 percent of the total weight of thebioactive topical formulation.
 7. A bioactive composition comprising anisolated bioactive fraction, wherein said isolated bioactive fraction isproduced by a process comprising: (i) providing fresh biomass of aCamellia sinensis plant, wherein said fresh biomass comprises freshCamellia sinensis plant biomass that has not undergone conventional teaprocessing; (ii) separating the fresh biomass of the Camellia sinensisplant into cell juice and a cell walls component, wherein saidseparating comprises grinding, macerating, and pressing the freshbiomass to separate the fresh biomass into the cell juice and the cellwalls component; (iii) treating the cell juice under conditionseffective to yield a bioactive fraction, said conditions comprisingsubjecting the cell juice to microwave treatment to induce coagulation,thereby yielding a coagulated membrane fraction and a non-coagulatedportion of the cell juice, separating the coagulated membrane fractionfrom the non-coagulated portion of the cell juice, thereby yielding acoagulated membrane fraction; and (iv) extracting the membrane fractionwith dimethyl sulfoxide, thereby yielding a membrane fraction extract,wherein the bioactive fraction is the membrane fraction extract; andwherein said membrane fraction extract has a catechin content profilecomprising: between about 1.7 and about 3.3 milligrams of(−)-epigallocatechin per gram of dry matter of the membrane fractionextract, between about 6.1 and about 10.2 milligrams of (+)-catechin pergram of dry matter of the membrane fraction extract, between about 0.3and about 1.1 milligrams of (−)-epicatechin per gram of dry matter ofthe membrane fraction extract, between about 6.2 and about 12.5milligrams of (−)-epigallocatechin gallate per gram of dry matter of themembrane fraction extract, between about 0.007 and about 0.03 milligramsof (−)-gallocatechin gallate per gram of dry matter of the membranefraction extract, and between about 1.3 and about 3.3 milligrams of(−)-epicatechin gallate per gram of dry matter of the membrane fractionextract.
 8. The bioactive composition according to claim 7, furthercomprising a stabilizing agent.
 9. The bioactive composition accordingto claim 8, wherein the said stabilizing agent is selected from thegroup consisting of an emulsifier, a preservative, an antioxidant, apolymer matrix, and mixtures thereof.
 10. A bioactive topicalformulation suitable for topical application to a mammal, said bioactivetopical formulation comprising: a topically effective amount of thebioactive composition according to claim 7 and a topically acceptablecarrier.
 11. The bioactive topical formulation according to claim 10,wherein the topically acceptable carrier is selected from the groupconsisting of a hydrophilic cream base, a hydrophilic lotion base, ahydrophilic surfactant base, a hydrophilic gel base, a hydrophilicsolution base, a hydrophobic cream base, a hydrophobic lotion base, ahydrophobic surfactant base, a hydrophobic gel base, and a hydrophobicsolution base.
 12. The bioactive topical formulation according to claim10, wherein the bioactive composition is present in an amount rangingfrom between about 0.001 percent and about 90 percent of the totalweight of the bioactive topical formulation.